IHC and ICC staining using single and multiple labels

Video Transcript

• 00:01 - 00:09: Hi everyone, welcome to this Abcam webinar entitled IHC and ICC staining techniques using single and multiple labels.
• 00:09 - 00:18: As the title suggests, I’m going to talk you through all of the important factors and steps that must be considered when performing both single and multiple label immunostaining.
• 00:19 - 00:31: Before we begin, it is worth noticing that a lot of the important aspects of designing IHC and ICC experiments were covered in the webinar Optimising IHC and ICC Results Through Careful Experimental Design.
• 00:31 - 00:34: A video of this can be found in the Abcam blog.
• 00:34 - 00:41: Today’s webinar focuses more on the practical aspects of performing immunostaining both with single and multiple labels.
• 00:41 - 00:47: However, for completeness, certain care experimental design steps will be reiterated where appropriate.
• 00:48 - 00:53: Okay, so we’re going to work through and discuss the following in detail.
• 00:53 - 01:00: Firstly, there’s IHC and ICC using a single reporter label, both day 1 and day 2 of the staining protocol.
• 01:00 - 01:09: Day 1 will consist of antigen retrieval, both heat induced and enzymatic, buffers and detergents, blocking buffers and finally primary antibody incubation.
• 01:09 - 01:22: Day 2 will cover quenching endogenous peroxidases, briefly touch on detection systems, discuss whether to use an enzymatic fluorescent reporter label and the various chromogens that are available for enzymatic detection.
• 01:22 - 01:27: We will then go on to talk about tinctural counterstains before finally discussing mounting.
• 01:27 - 01:32: In case you were wondering, I will talk about fluorescent counterstains in the multiple staining section.
• 01:33 - 01:44: In the second part of the webinar, we will look at IHC and ICC using multiple reporter labels, by beginning with how fluorescence works before moving on to combinations of fluorescent reporter labels.
• 01:44 - 01:54: We will then talk about fluorescence compared to enzyme reporter labels, followed by simultaneous staining, then sequential staining, finishing with some discussion about controls.
• 01:55 - 02:06: Firstly, we will go through a generic IHC or ICC protocol suitable for use with a labelled secondary antibody, polymer or avidin-binding complex.
• 02:06 - 02:13: As you will have learned in the past webinar, there are numerous variations on each step, depending on your experimental design.
• 02:13 - 02:20: I will talk you through each protocol step and, when needed, I will discuss any common experimental design variations.
• 02:21 - 02:26: The focus for the last part of the webinar will be the design of experiments using multiple labels.
• 02:30 - 02:38: We will go into these in much more detail, but it is worth just quickly talking through the individual steps in the protocol, just to give you an idea of what is to come.
• 02:38 - 02:46: First of all, on day one, any pre-treatments are performed, such as de-waxing of paraffin sections and antigen retrieval.
• 02:46 - 02:55: There is then a wash stage, followed by blocking nonspecific protein-protein interactions, or in other words, nonspecific staining, after which there is another wash.
• 02:55 - 03:00: The primary antibody is then incubated on the specimen overnight at 4 degrees centigrade.
• 03:03 - 03:13: We then go on to day two, and the first job is to wash off the primary antibody before blocking any endogenous peroxidases, if indeed you are using a peroxidase enzyme reporter label.
• 03:14 - 03:21: After this, it is time to start introducing the detection system and allowing it to incubate before washing it off.
• 03:21 - 03:31: If the detection system you are using is ABC-based, you would add the ABC complex at this point, let it incubate and wash it off, but if not, you would skip this step.
• 03:31 - 03:39: Similarly, if you are using an enzyme reporter label, you would then incubate in chromogen before washing it off, but if not, you would also skip this step.
• 03:40 - 03:46: You would also block any endogenous phosphatases at this point, if you are using a phosphatase enzyme reporter label.
• 03:46 - 03:50: Any counterstains would then be applied before washing off the excess.
• 03:50 - 04:02: If you are using an enzyme reporter label and chromogen combination that is not alcohol soluble, you would next incorporate a dehydrator clear and mount procedure, but once again, if not, you would also skip this step.
• 04:03 - 04:12: Then finally, whatever happens, you are going to want to mount your stained specimen in some way in order to help preserve it before, during and after microscopic analysis.
• 04:14 - 04:25: Let’s now discuss the first step of the protocol in a little more detail, which is concerned with performing any required specimen pre-treatments, such as de-waxing of tissue sections and antigen retrieval.
• 04:26 - 04:37: Antigen, or epitope, retrieval serves to break the methylene bridges formed during aldehyde fixation, allowing antibodies access to certain fixation-sensitive antigens.
• 04:37 - 04:46: Antigen retrieval is therefore only necessary on specimens that have undergone aldehyde fixation, and only then if a fixation-sensitive antigen is being demonstrated.
• 04:46 - 04:51: All specimens should be on coated slides to help prevent disassociation.
• 04:52 - 05:00: Both frozen and paraffin sections are used to survive antigen retrieval if mounted on coated slides, and if they are particularly friable, such as adipose tissue.
• 05:01 - 05:13: There are two common methods of antigen retrieval, heat-induced, or HIR here for short, which involves the use of a suitable buffer, such as trisodium citrate at pH 6 or EDTA at pH 9.
• 05:14 - 05:22: The other is enzymatic, which involves the use of a suitable enzyme solution, such as trypsin, pepsin, pronase, or proteinase K.
• 05:22 - 05:30: Both methods can be incorporated into automated systems, either as part of an immunostaining platform or a standalone antigen retrieval vessel,
• 05:30 - 05:38: but if done manually, heat-induced antigen retrieval is commonly performed in a pressure cooker or microwave, and enzymatic in a water bath.
• 05:39 - 05:50: The following antigen retrieval protocols are generic, concentrating on the above methods using trisodium citrate at pH 6 for heat-induced antigen retrieval and chymotrypsin for enzymatic.
• 05:50 - 05:54: However, please remember that there is no universal antigen retrieval solution.
• 05:54 - 06:02: For each antigen, the most appropriate antigen retrieval solution, or enzyme, pH, method, and duration must be established.
• 06:03 - 06:15: So, for performing heat-induced antigen retrieval, you begin by preparing the trisodium citrate buffer by mixing together 5.88 grams of trisodium citrate,
• 06:15 - 06:23: 44 millilitres of 0.2 molar hydrochloric acid, and 1,956 millilitres of ultra-pure water.
• 06:23 - 06:34: This would then be adjusted to pH 6 with 1 molar sodium hydroxide and hydrochloric acid, before being poured into the pressure cooker, using enough volume to cover the slides by about a centimetre.
• 06:34 - 06:41: The pressure cooker itself would then be put onto a hot plate, and the lid placed on top, and by this I mean don’t secure it down.
• 06:41 - 06:44: The hot plate would then be turned on to bring the buffer to the boil.
• 06:45 - 06:53: While waiting for the pressure cooker to come to the boil, de-wax and rehydrate any paraffin sections by placing them in a suitable rack,
• 06:53 - 07:01: and then three changes of xylene for three minutes each, followed by three changes of alcohol for three minutes each, followed by cold running domestic supply water.
• 07:01 - 07:05: Keep them in the running water until the pressure cooker comes to the boil.
• 07:05 - 07:12: With de-waxing, the xylene removes the wax, the alcohol then removes the xylene, and finally, the water removes the alcohol.
• 07:12 - 07:23: In other words, the slides are going from an organic to an aqueous phase, using alcohol as an intermediate phase, since both organic and aqueous liquids are miscible in alcohol.
• 07:23 - 07:30: Once the buffer is boiling, transfer the slides from the running water to the pressure cooker, taking care with the hot solution and steam.
• 07:30 - 07:38: Secure the pressure cooker lid following the manufacturer’s instructions, and once the cooker has reached full pressure, set a timer for three minutes.
• 07:39 - 07:44: When the three minutes has elapsed, turn off the hot plate and place the pressure cooker in an empty sink.
• 07:44 - 07:51: Activate the pressure release valve and run cold domestic supply water over the cooker to aid depressurisation.
• 07:52 - 08:00: Once depressurised, open the lid and run cold domestic supply water into the cooker for ten minutes, again taking care with the hot solution and steam.
• 08:00 - 08:04: You are now ready to continue with the immunostaining protocol.
• 08:05 - 08:12: Please note that this protocol can be modified with regards to buffer use, pH and duration of antigen retrieval.
• 08:12 - 08:15: Three minutes duration is only a suggested starting point.
• 08:15 - 08:22: Less than three minutes may leave the antigens under-retrieved, and more than this may leave them over-retrieved,
• 08:22 - 08:28: which may cause false or background staining and may increase section disassociation from the slides.
• 08:29 - 08:39: Optimisation is therefore advised, retrieving slides to 1, 3, 5, 10 and 20 minutes before being immunochemically stained.
• 08:39 - 08:46: This protocol can easily be modified for use with a microwave, but always begin retrieval timings from when the buffer is boiling.
• 08:46 - 08:53: If you choose to use a microwave, it is advisable to use a scientific one with a temperature probe and a stirring function
• 08:53 - 09:01: in order to prevent the retrieval solution from boiling vigorously, thus aiding section disassociation and to eliminate cold spots.
• 09:01 - 09:11: Finally, and probably most importantly, please follow the manufacturer’s instructions for the safe use of a particular pressure cooker or microwave that you have in your lab.
• 09:11 - 09:16: Please also follow health and safety guidelines for any of the chemicals that you are using.
• 09:17 - 09:21: Let us now look at enzymatic antigen retrieval protocol.
• 09:21 - 09:26: Begin by setting a water bath to 37 degrees centigrade.
• 09:26 - 09:36: Next, obtain two troughs large enough to accommodate the rack slides and add a sufficient amount of ultra-pure water to each trough to cover the slides.
• 09:36 - 09:43: Place the troughs into the water bath and allow the ultra-pure water to warm to 37 degrees centigrade.
• 09:43 - 09:54: De-wax and rehydrate any paraffin sections as outlined in the heat-induced antigen retrieval protocol and place them into one trough of ultra-pure water at 37 degrees C to warm.
• 09:54 - 10:06: Remove the other trough of ultra-pure water and to this dissolve 0.1 grams of calcium chloride and 0.1 grams of chymotrypsin per 100 millilitres of ultra-pure water,
• 10:06 - 10:10: using a magnetic stirrer to ensure that all reagents are properly dissolved.
• 10:10 - 10:19: Once dissolved, bring the solution to pH 7.8 using 1 molar sodium hydroxide and hydrochloric acid before returning the trough to the water bath.
• 10:19 - 10:24: Allow this enzyme solution to reheat to 37 degrees centigrade.
• 10:24 - 10:32: Transfer the warm slides into the enzyme solution for 20 minutes and then remove them and place them in cold running domestic supply water for 3 minutes.
• 10:32 - 10:37: You are now ready to continue with the immunostaining protocol.
• 10:37 - 10:51: Chymotrypsin features in this protocol, but any enzyme can be used, just ensure to adjust the Vmax temperature and pH accordingly, pH 7.8 and 37 degrees centigrade being those for chymotrypsin.
• 10:51 - 10:58: Pre-warming the slides in ultra-pure water avoids reducing the temperature of the enzyme solution when they are placed within it.
• 10:59 - 11:06: Ensure to make up the enzyme solution fresh and use it straight away, since any delay could affect its proteolytic properties.
• 11:06 - 11:12: Similarly, ensure that the enzyme solution reaches its Vmax temperature before introducing the slides.
• 11:12 - 11:20: As with heat-induced antigen retrieval, less than 20 minutes may leave the antigens under-retrieved and more than this may leave them over-retrieved,
• 11:21 - 11:27: which may cause false or background staining and may increase section disassociation from the slides.
• 11:27 - 11:36: Optimisation is again therefore advised, retrieving slides for 5, 10, 15, 20 and 25 minutes before being immunochemically stained.
• 11:36 - 11:43: Most importantly, please also follow health and safety guidelines for any of the chemicals that you are using.
• 11:44 - 11:50: Okay, we have now completed the first step in our protocol and we are about to move on to the second,
• 11:50 - 11:55: which is the first of many washes in buffer containing detergent.
• 11:58 - 12:02: A common question regarding buffers is generally what is best to use.
• 12:02 - 12:09: Well again, there isn’t one universal buffer, you have to use the one most suitable according to your experimental design parameters.
• 12:09 - 12:16: As a rough guide, I would personally recommend TBS containing 0.5% Triton X-100 for tissue sections
• 12:16 - 12:21: and PBS containing 0.1% Tween for cytological preparations.
• 12:22 - 12:25: TBS is of higher ionic strength than PBS.
• 12:25 - 12:30: TBS therefore helps to give cleaner background staining but is often unsuitable for cytological specimens
• 12:30 - 12:34: since it can osmotically cause cells to lyse.
• 12:34 - 12:39: Detergents, or more specifically in this case surfactants, such as Tween and Triton,
• 12:39 - 12:45: improve antibody penetration by removing lipids from cell membranes, therefore permeabilising them.
• 12:45 - 12:50: The demonstration of most antigens certainly benefits from detergent being present in the buffer.
• 12:50 - 12:55: However, please be aware that some cell membrane proteins may be stripped out using a detergent,
• 12:55 - 12:58: so it is best to omit it in such cases.
• 12:58 - 13:02: Detergents reduce surface tension, helping reagents spread out over the specimen
• 13:02 - 13:08: and are also considered to dissolve Fc receptors in frozen sections, reducing background staining.
• 13:09 - 13:14: Triton is more aggressive than Tween, making Tween more suitable for cytological specimens
• 13:14 - 13:16: since they tend to be more friable.
• 13:16 - 13:21: I have found that an addition of 0.1% Tween in buffers used for cytological preparations
• 13:21 - 13:25: gives a gentler degree of permeabilisation than Triton.
• 13:25 - 13:28: If more rigorous permeabilisation is required,
• 13:28 - 13:32: 10 minutes incubation in 4 millimolar sodium deoxycholate can be used
• 13:32 - 13:37: between protocol steps 1 and 2, with an additional buffer wash before step 2.
• 13:37 - 13:41: The concentration of detergent can simply be increased also,
• 13:41 - 13:45: but pay attention to the potentially negative effects as previously described.
• 13:45 - 13:49: Incidentally, if you are using a phosphatase label,
• 13:49 - 13:54: avoid making it up in PBS, since the phosphates in the buffer will inhibit it.
• 13:54 - 13:59: After the first wash, we now move on to the blocking step.
• 13:59 - 14:03: Blocking buffer helps prevent unwanted background staining
• 14:03 - 14:07: that could mask positive staining or lead to false positive results.
• 14:07 - 14:11: This is achieved by incubating in buffer containing detergent,
• 14:11 - 14:15: which can be used to prevent unwanted background staining.
• 14:16 - 14:20: This is achieved by incubating in buffer containing detergent,
• 14:20 - 14:26: plus 10% normal serum, 1% BSA and 0.3 molar glycine for 2 hours at room temperature.
• 14:26 - 14:31: Use normal serum from the species used to raise a secondary antibody.
• 14:31 - 14:36: Any endogenous immunoglobulins in the specimen that have affinity with the secondary antibody
• 14:36 - 14:39: will be preabsorbed by the immunoglobulins in the serum.
• 14:39 - 14:43: BSA serves a similar purpose by saturating any proteins
• 14:43 - 14:47: that will react non-specifically with other proteins, such as antibodies.
• 14:47 - 14:52: In aldehyde-fixed specimens, glycine binds to any free aldehyde groups,
• 14:52 - 14:56: reducing the incidence of hydrophobic protein-protein interactions.
• 14:56 - 15:01: The image on this slide shows an example of what can happen when the wrong serum is used.
• 15:01 - 15:05: Serum from the same species that the secondary was raised against has been used,
• 15:05 - 15:09: and opposed to serum from the same species from which the secondary was raised.
• 15:09 - 15:14: Hence a secondary antibody, in red, binding non-specifically over the entire specimen.
• 15:18 - 15:21: We have now performed blocking, and after another wash step,
• 15:21 - 15:24: we will move on to addition of the primary antibody.
• 15:27 - 15:29: The primary antibody is applied to the specimen,
• 15:29 - 15:34: optimally diluted in buffer containing detergent, plus 1% BSA.
• 15:34 - 15:38: The primary antibody will target the epitope it is raised against.
• 15:39 - 15:43: Ensure that the primary antibody is raised in a different species than the specimen,
• 15:43 - 15:46: in order to reduce background staining from the secondary antibody,
• 15:46 - 15:48: binding to endogenous immunoglobulin.
• 15:48 - 15:52: This isn’t really a problem when using cytological specimens,
• 15:52 - 15:54: unless you are staining cells of a B cell lineage,
• 15:54 - 15:57: which is unlikely, since they are non-adherent by nature.
• 16:00 - 16:03: Once the primary antibody has been added to the specimen,
• 16:03 - 16:07: the next step is to incubate it overnight at 4 degrees centigrade.
• 16:09 - 16:14: Lower affinity antibodies are allowed more time to bind in an overnight incubation.
• 16:15 - 16:18: Overbinding isn’t an issue, since once the antigens are saturated,
• 16:18 - 16:20: no further binding can occur.
• 16:21 - 16:25: The reduced temperature helps to reduce background staining by increasing reaction times
• 16:25 - 16:29: and decreasing the incidence of non-specific protein-protein interactions.
• 16:30 - 16:35: The effects of this can be enhanced by gentle specimen agitation from an orbital shaker.
• 16:35 - 16:40: An overnight incubation is not usually essential, but has the advantages outlined above.
• 16:41 - 16:44: You will have hopefully already performed an optimisation experiment
• 16:44 - 16:47: with regards to the optimal primary antibody dilution.
• 16:48 - 16:52: After all of that, congratulations, day 1 is finished.
• 16:52 - 16:56: Time to go home and relax to arrive all fresh and ready to tackle day 2.
• 16:57 - 17:00: Our primary antibodies have been incubated overnight
• 17:00 - 17:03: and we are now back in the lab to do day 2 of our immunostaining protocol.
• 17:04 - 17:08: This begins by giving our slides another wash to remove the primary antibody
• 17:08 - 17:11: before going on to blocking endogenous peroxidases,
• 17:11 - 17:14: if we are using a peroxidase enzyme reporter label, that is.
• 17:14 - 17:19: If not, you just skip steps 2 and 3, going straight on to step 4.
• 17:23 - 17:26: Blocking endogenous peroxidases only applies to detection systems
• 17:26 - 17:30: that use a horseradish peroxidase, or HRP, reporter label.
• 17:30 - 17:33: Red blood cells contain endogenous peroxidases,
• 17:33 - 17:35: which if not quenched with hydrogen peroxide,
• 17:35 - 17:39: will react with the chromogen alongside the peroxidase reporter label,
• 17:39 - 17:41: producing false positive staining.
• 17:41 - 17:46: Incubating paraffin-embedded specimens in 1.6% hydrogen peroxide
• 17:46 - 17:50: made up in buffer-containing surfactant for 30 minutes at room temperature
• 17:50 - 17:54: or 5 minutes for a frozen section of cytological preparation
• 17:54 - 17:57: adequately blocks endogenous peroxidase activity
• 17:57 - 18:00: without having a detrimental effect on tissue epitopes.
• 18:00 - 18:05: That is why endogenous peroxidase quenching is done after primary antibody binding
• 18:05 - 18:08: in order to ensure that this isn’t an issue.
• 18:09 - 18:12: You should only use fresh hydrogen peroxide
• 18:12 - 18:15: since it quickly breaks down into water and oxygen at room temperature,
• 18:15 - 18:18: making it ineffective at quenching.
• 18:18 - 18:23: It is good practice to store hydrogen peroxide frozen and thawed directly before use.
• 18:23 - 18:28: Aside, when staining tissues high in endogenous peroxidases, such as spleen,
• 18:28 - 18:31: it is sometimes best to use a non-peroxidase reporter label,
• 18:31 - 18:34: which is alkaline phosphatase, or AP.
• 18:34 - 18:38: Quenching of endogenous phosphatases will be discussed later.
• 18:40 - 18:42: The next step, step four,
• 18:42 - 18:46: is to begin incubating the specimen with the chosen detection system.
• 18:46 - 18:50: This protocol focuses on three popular systems,
• 18:51 - 18:55: those being a directly reporter label conjugated primary antibody,
• 18:55 - 18:59: a biotinylated secondary antibody, if an ABC,
• 18:59 - 19:03: avidin-binding complex, or a secondary antibody reporter label complex
• 19:03 - 19:07: in the form of a dextran polymer or compact polymer.
• 19:07 - 19:10: I went into how each of these systems work
• 19:10 - 19:13: and the subject of signal amplification in my last webinar,
• 19:13 - 19:15: including the pros and cons of each,
• 19:15 - 19:17: so I’m not going to dwell on this here,
• 19:17 - 19:19: but you can check it out on the Abcam blog.
• 19:20 - 19:22: Please note that if using an ABC system,
• 19:22 - 19:25: there will be an additional step, step six,
• 19:25 - 19:28: which I will talk about in more detail later,
• 19:28 - 19:30: where the actual ABC complex is added.
• 19:30 - 19:32: So at this stage, step four,
• 19:32 - 19:36: it will just be the biotinylated secondary antibody that is added.
• 19:37 - 19:40: In general, ensure that all of the detection system reagents
• 19:40 - 19:45: are optimally diluted in buffer-containing surfactant plus 1% BSA
• 19:45 - 19:48: and incubated on the specimen for one hour at room temperature.
• 19:51 - 19:53: This is a poignant place to discuss
• 19:53 - 19:57: whether to use an enzyme reporter label or a fluorescent one.
• 19:57 - 20:00: A very common dilemma is whether to use a fluorescent
• 20:00 - 20:03: or enzyme reporter label in a particular experiment.
• 20:03 - 20:07: Enzymatic reporter labels are commonly used on tissue sections
• 20:07 - 20:11: and fluorescent reporter labels are commonly used on frozen tissue sections
• 20:11 - 20:13: and cytological preparations.
• 20:13 - 20:15: However, this is not absolute.
• 20:15 - 20:20: The detection system should be tailored to suit the immunostaining experiment.
• 20:20 - 20:23: The main benefit of fluorescence over enzymatic
• 20:23 - 20:26: is that all of the fluorescent channels can easily be viewed separately
• 20:26 - 20:29: and then merged to form a pseudo-coloured image.
• 20:29 - 20:32: It is therefore easy to see signal co-localisation
• 20:32 - 20:36: between a fluorescent counter stain and that of the detection system
• 20:36 - 20:39: without any specialised spectral or mixing software.
• 20:40 - 20:44: Weak signals from the primary antibody can be also observed in isolation
• 20:44 - 20:47: without any interference from other signals.
• 20:49 - 20:52: Often, tissue sections display autofluorescence
• 20:52 - 20:56: due to some tissue components being naturally fluorescent, such as collagen.
• 20:56 - 21:00: Formaldehyde fixation also increases the degree of autofluorescence.
• 21:00 - 21:04: If strong enough, it can mask the signal from fluorescent reporter labels
• 21:04 - 21:07: making results interpretation difficult.
• 21:08 - 21:12: Enzymatic detection is therefore more appropriate for tissue sections.
• 21:12 - 21:15: Cytological preparations and frozen sections
• 21:15 - 21:19: are commonly not exposed to formaldehyde for long enough to exacerbate autofluorescence
• 21:19 - 21:24: and cytological preparations often do not possess such naturally fluorescent components.
• 21:29 - 21:32: Our detection system is now incubated for an hour
• 21:32 - 21:34: and we wash it off with a buffer rinse.
• 21:34 - 21:39: If we are using an ABC system, it is at this stage that it will be incubated on the specimen.
• 21:39 - 21:42: Make this up according to the manufacturer’s instructions.
• 21:42 - 21:45: The ABC system is usually provided as avidin alone
• 21:45 - 21:48: with the biotin label complex in a separate container.
• 21:48 - 21:51: End users must mix both accordingly
• 21:51 - 21:54: and wait for at least 30 minutes for complex formation
• 21:54 - 21:56: before adding it to the specimen
• 21:56 - 22:00: or it will not function to its full capability as a detection reagent.
• 22:00 - 22:03: However, if you are not using an ABC system
• 22:03 - 22:07: you skip steps 6 and 7 and proceed directly to step 8.
• 22:09 - 22:12: If you are using an enzyme reporter label
• 22:12 - 22:14: you are now ready to add the chromogen.
• 22:16 - 22:19: Reporter labels that are enzymatic in nature
• 22:19 - 22:23: produce a stable coloured precipitate at the site of primary antibody binding
• 22:23 - 22:25: when exposed to a suitable chromogen.
• 22:25 - 22:29: Among several, the two most popular enzyme labels for immunochemistry
• 22:29 - 22:32: are horseradish peroxidase and alkaline phosphatase.
• 22:32 - 22:35: Each of these has commonly used chromogens
• 22:35 - 22:39: namely AEC, DAB and DAB containing nickel for HRP
• 22:39 - 22:43: and Fast Blue, Fast Red and Naphthol AS for AP.
• 22:43 - 22:45: As you can see from the table
• 22:45 - 22:48: the different enzyme and chromogen combinations
• 22:48 - 22:52: produce a different coloured precipitate at the site of antibody binding.
• 22:52 - 22:55: Also note that some of the precipitates are alcohol soluble
• 22:55 - 22:58: which has an important significance when mounting
• 22:58 - 23:00: which I will come on to later.
• 23:02 - 23:06: Ensure that all chromogens are made up according to the manufacturer’s instructions
• 23:06 - 23:09: and incubate them on the specimen according to manufacturer’s guidelines.
• 23:09 - 23:14: Typically for DAB, 10 minutes at room temperature generally gives good results.
• 23:14 - 23:18: Always observe chromogen expiry dates and storage conditions.
• 23:18 - 23:23: HRP and hydrogen peroxide form a complex in the presence of DAB chromogen
• 23:23 - 23:28: with HRP catalysing the breakdown of hydrogen peroxide into water and oxygen.
• 23:28 - 23:34: DAB is oxidised during this process and provides electrons to drive the reaction.
• 23:34 - 23:40: It is the oxidised DAB that forms the black-brown coloured precipitate at the site of the reaction.
• 23:40 - 23:44: Hydrogen peroxide therefore plays a key role in this reaction
• 23:44 - 23:47: and since it quickly breaks down into water and oxygen at room temperature
• 23:47 - 23:51: DAB kits that have passed their expiry date may be ineffective.
• 23:52 - 23:57: Appropriate COSHH guidelines should be observed regarding the storage, use, handling
• 23:57 - 23:59: and disposal of any laboratory reagents.
• 24:00 - 24:03: If using an alkaline phosphatase reporter label
• 24:03 - 24:08: add 0.24 mg to ml levamisole to the working chromogen solution.
• 24:08 - 24:13: Levamisole quenches endogenous phosphatase activity reducing unwanted background staining
• 24:13 - 24:16: although not in placenta or small intestine.
• 24:17 - 24:22: Don’t worry, the phosphatase reporter label itself will not be affected by the levamisole.
• 24:25 - 24:28: After washing off the chromogen and running domestic supply water
• 24:28 - 24:31: we can now apply a suitable counterstain.
• 24:33 - 24:36: Counterstains add colour contrast to the cells or tissues
• 24:36 - 24:42: by staining certain cellular structures thus helping to define the localisation of the immunostaining.
• 24:43 - 24:47: They could be tinctural or fluorescent in nature complementing the detection system
• 24:47 - 24:50: used to visualise the primary antibody.
• 24:50 - 24:54: For enzyme detection systems, tinctural nuclear counterstains are commonly used.
• 24:54 - 24:58: I’ll talk more about fluorescent counterstains in the second section of this webinar.
• 25:00 - 25:05: The most common nuclear counterstain used when employing an enzyme chromogen detection system
• 25:05 - 25:06: is haematoxylin.
• 25:07 - 25:10: Haematoxylins are available in numerous formulations
• 25:10 - 25:15: identified by the type of mordant used and whether they stain progressively or regressively.
• 25:15 - 25:22: Haematoxylin stains cell nuclei in various shades of purple or blue according to the type used.
• 25:22 - 25:28: Haematoxylin or specifically its oxidation product haematin is anionic
• 25:28 - 25:30: and therefore has little affinity for DNA.
• 25:30 - 25:36: Mordants are iron salts which combine with haematin creating a positively charged dimordant complex
• 25:36 - 25:39: with the ability to bind to anionic chromatin.
• 25:39 - 25:45: Alum or aluminium mordant haematoxylins can be used progressively or regressively.
• 25:45 - 25:49: Progressive haematoxylins, for example maize, gills or kerases,
• 25:49 - 25:54: can be applied to tissues or cells until the desired degree of nuclear staining is observed.
• 25:55 - 26:01: Aggressive haematoxylins such as Harris are applied to tissues until overstaining is observed
• 26:01 - 26:07: before having a proportion of the excess staining removed by immersion in acid such as 1% acid alcohol.
• 26:07 - 26:10: This process is called differentiation.
• 26:10 - 26:16: This makes progressive haematoxylin simpler to use than regressive due to the omission of the differentiation step
• 26:16 - 26:21: and the subsequent compatibility with alcohol soluble enzyme substrate end products
• 26:21 - 26:25: such as that produced by the reaction of HRP and AEC.
• 26:25 - 26:31: All haematoxylins, both progressive and regressive, are blued once the desired level of staining has been achieved.
• 26:31 - 26:36: Haematoxylin stains nuclei red and drastic under acidic conditions.
• 26:36 - 26:42: However, in an alkaline environment, haematoxylin turns a pleasing blue-purple colour.
• 26:43 - 26:47: Running domestic supply water is commonly used for this purpose.
• 26:47 - 26:52: It usually has sufficient alkalinity to achieve this, especially in hard water areas.
• 26:52 - 26:59: In soft water areas, an alkaline solution can be used for blueing such as 0.05% ammonia.
• 27:00 - 27:06: Other commonly used tinctural nuclear counterstains are light green, Fast Red, toluidine blue and methylene blue.
• 27:06 - 27:10: Staining nuclei are either green, red or blue respectively.
• 27:10 - 27:14: One important consideration when using a nuclear tinctural counterstain
• 27:14 - 27:18: is not to make the staining too intense if you are demonstrating a nuclear antigen
• 27:18 - 27:23: since the counterstain can potentially mask a positive signal from the detection system.
• 27:23 - 27:27: Let’s now discuss specimen preparation for microscopic analysis.
• 27:28 - 27:33: It is essential to prepare the immunostained specimen in order to preserve it while being imaged
• 27:33 - 27:36: during long-term storage and to enhance image quality.
• 27:36 - 27:40: This typically involves placing a glass coverslip over the specimen,
• 27:40 - 27:44: securing it in place with a suitable adhesive known as mounting media.
• 27:44 - 27:48: Mounting media can be either aqueous, superglued or non-glued.
• 27:49 - 27:54: Organic mounting media tend to set hard, allowing the glass coverslip to remain securely in place.
• 27:57 - 28:01: Refractive indexes are better with organic mounting media such as DPX,
• 28:01 - 28:05: giving a much sharper, crisper image down the microscope.
• 28:05 - 28:09: However, ensuring that the glass coverslip is securely in place
• 28:09 - 28:13: can be difficult to achieve with non-refractive indexes.
• 28:14 - 28:18: For example, the reaction of peroxidase and AEC is alcohol soluble,
• 28:18 - 28:23: so will disappear during the dehydrating and clearing process if an organic mounting media is used.
• 28:23 - 28:26: Fluorescent labels require aqueous mounting media.
• 28:26 - 28:31: 10% glycerol can be used, but commercially available media containing anti-fade reagents
• 28:31 - 28:34: such as glycerol can be used.
• 28:34 - 28:38: For example, glycerol can be used in anti-fade reagents,
• 28:38 - 28:44: 10% glycerol can be used, but commercially available media containing anti-fade reagents are superior.
• 28:44 - 28:49: When using a fluorescent label, it is advisable to microscopically observe the mounting media
• 28:49 - 28:54: on a blank coverslip to ensure that it does not produce any autofluorescence.
• 28:58 - 29:03: I’m happy to say that our immunostaining specimen is now ready to be analysed down the microscope.
• 29:03 - 29:06: Microscopic analysis is a complete webinar on its own,
• 29:06 - 29:10: especially to go into how to set up the microscope for optimal illumination
• 29:10 - 29:15: for both light microscopy and fluorescence, and so I won’t be going into that right now.
• 29:18 - 29:21: Right then, let’s now think about multiple staining.
• 29:24 - 29:27: The purpose of immunostaining using multiple reporter labels
• 29:27 - 29:32: is to simultaneously visualise the cellular localisation of two or more antigens
• 29:32 - 29:35: in the same cell or tissue section.
• 29:35 - 29:40: A different reporter label is used for each antigen in order to distinguish them apart.
• 29:41 - 29:47: Antigens may be co-localised, meaning within the same cellular compartment of any given cell, or separate.
• 29:47 - 29:50: When two or more antigens are co-localised,
• 29:50 - 29:55: the colour of the separate reporter labels will mix to produce a new colour.
• 29:55 - 29:58: Both fluorescent and enzyme reporter labels can be used,
• 29:58 - 30:02: but these need to be carefully selected to ensure that they do not interfere with each other
• 30:02 - 30:05: and that they are easily inter-distinguishable.
• 30:06 - 30:12: Similarly, the detection system reagents must not cross-react or interfere with each other in any way.
• 30:12 - 30:16: This webinar will concentrate on fluorescence multistaining.
• 30:16 - 30:21: That way, there’s always the potential for another separate webinar for enzymatic later.
• 30:23 - 30:27: Before we go on, we will first consider how fluorescence works.
• 30:28 - 30:32: A fluorescent molecule has the ability to absorb light of a specific wavelength
• 30:32 - 30:35: and to re-emit light at a longer wavelength.
• 30:35 - 30:38: It loses energy in order to achieve this,
• 30:38 - 30:41: by interacting with its environment prior to the emission of fluorescence,
• 30:41 - 30:44: a phenomenon called internal conversion,
• 30:44 - 30:47: which is the loss of energy in the absence of light emission.
• 30:47 - 30:52: Electrons can exist either at a ground state or resting state called S0,
• 30:52 - 30:56: or in excited states of higher energy called S1 and S2.
• 30:56 - 30:58: At each of these electronic states,
• 30:58 - 31:04: fluorochromes can exist in a number of vibrational levels called 0, 1 and 2.
• 31:04 - 31:10: There is not enough energy at room temperature to populate the excited electronic states of S1 and S2,
• 31:10 - 31:13: or the higher vibrational levels of the ground state.
• 31:13 - 31:18: Therefore, absorption generally occurs from molecules in the lowest vibrational energy state
• 31:18 - 31:20: and only in the presence of light.
• 31:20 - 31:26: Following absorption, a fluorochrome is usually excited to a higher vibrational level
• 31:26 - 31:28: of either S1 or S2,
• 31:28 - 31:31: before relaxing to the lowest vibrational level of S1,
• 31:31 - 31:34: thus completing the process of internal conversion.
• 31:34 - 31:38: This, combined with the emission of a photon, results in fluorescence.
• 31:38 - 31:43: A fluorochrome can repeat the excitation emission cycle many times
• 31:43 - 31:46: before excitation bleaches the fluorescent state.
• 31:46 - 31:50: However, some fluorophores photobleach more readily than others.
• 31:50 - 31:54: FITC, for example, can repeat the excitation emission process
• 31:54 - 31:58: approximately 30,000 times before photobleaching occurs.
• 31:58 - 32:03: Second generation fluorochromes, however, such as the Alexa Fluor range,
• 32:03 - 32:06: do not photobleach as rapidly and are generally brighter
• 32:06 - 32:09: since they have a higher extinction coefficient.
• 32:10 - 32:14: With this in mind, we will now discuss how to go about choosing combinations
• 32:14 - 32:18: of fluorescent reporter labels for a multicolor staining experiment.
• 32:23 - 32:27: Unless the fluorescent reporter labels are of the same color,
• 32:27 - 32:30: they cannot be used for a fluorescent staining experiment.
• 32:30 - 32:34: However, if the fluorescent reporter labels are of the same color,
• 32:34 - 32:37: they can be used for a fluorescent staining experiment.
• 32:38 - 32:42: Unless the appropriate spectral mixing software is available,
• 32:42 - 32:46: fluorochromes must be selected so that their emission spectra do not overlap.
• 32:47 - 32:51: This is critical, otherwise the emission of one fluorophore
• 32:51 - 32:54: may be detected in the filter set intended for another fluorophore.
• 32:54 - 32:58: This phenomenon is known as bleed-through, or cross-talk, or cross-over.
• 32:58 - 33:02: Bleed-through occurs due to the non-symmetrical spectral properties
• 33:02 - 33:05: of many fluorophores and associated wide bandwidths.
• 33:05 - 33:09: Consider the simultaneous spectral emission analysis of
• 33:09 - 33:14: Alexa Fluor 488 and Alexa Fluor 555 in the lower right image.
• 33:14 - 33:18: The maximum emission peaks are seemingly well defined and separated,
• 33:18 - 33:22: but at the maximum emission peak for Alexa Fluor 555,
• 33:22 - 33:26: there is still a significant overlap with that of Alexa Fluor 488.
• 33:26 - 33:32: This creates bleed-through of Alexa Fluor 488 into the Alexa Fluor 555 channel.
• 33:32 - 33:36: This same phenomenon can be seen in action in the top right-hand image
• 33:36 - 33:39: with Cy3 bleeding through into Texas Red.
• 33:41 - 33:45: The aim, therefore, is to minimise the area of emission overlap
• 33:45 - 33:48: by using another fluorophore of long enough emission wavelength
• 33:48 - 33:51: to minimise the overlap or to eliminate it.
• 33:51 - 33:56: If Alexa Fluor 594 were used in place of Alexa Fluor 555,
• 33:56 - 33:59: the bleed-through effect of Alexa Fluor 488 would be small enough
• 33:59 - 34:03: for both dyes to be used together in a multiple labelling experiment
• 34:03 - 34:09: since the emission profile of Alexa Fluor 594 sits further to the right of Alexa Fluor 488
• 34:09 - 34:14: than Alexa Fluor 555 since the area of emission overlap is smaller.
• 34:14 - 34:18: This is why 3 or 4 colour fluorescents using one fluorophore
• 34:18 - 34:21: from separate areas of the spectrum is so popular
• 34:21 - 34:26: since their absorption and emission spectra are adequately far enough apart
• 34:27 - 34:30: to allow no or very little bleed-through.
• 34:32 - 34:36: For instance, a traditional dye combination of DAPI for blue,
• 34:36 - 34:41: FITC for green, TRITC for yellow and Cy5 for red is commonly used.
• 34:41 - 34:45: An equivalent second generation dye combination of this would be
• 34:45 - 34:51: DAPI for blue, Alexa Fluor 488 for green, Alexa Fluor 555 for yellow
• 34:51 - 34:53: and Alexa Fluor 647 for red.
• 34:54 - 34:57: It is worth mentioning at this stage that not every fluorescent signal
• 34:57 - 34:59: needs to come from a conjugated antibody.
• 34:59 - 35:02: It can be from a nuclear counterstain such as DAPI,
• 35:02 - 35:06: from an organelle-specific stain such as Mito Tracker
• 35:06 - 35:09: or from a cell membrane counterstain such as wheat germ agglutinin
• 35:09 - 35:11: conjugated to a fluorescent label.
• 35:12 - 35:15: Carefully selected microscope absorption and emission filters
• 35:15 - 35:18: can drastically help to reduce bleed-through.
• 35:18 - 35:21: Indeed, it is the filter sets on the user’s microscope
• 35:21 - 35:23: that will ultimately dictate the fluorophores
• 35:23 - 35:25: that can be successfully used.
• 35:29 - 35:32: There are several steps that can be taken when designing
• 35:32 - 35:35: a multi-labelling experiment using fluorescent reporter labels
• 35:35 - 35:38: to help minimise or eliminate bleed-through.
• 35:39 - 35:43: Firstly, use a spectral analyser program to observe
• 35:43 - 35:47: the absorption and emission spectra of your potential fluorophores
• 35:48 - 35:51: alongside the characteristics of your microscope filter set
• 35:51 - 35:54: to assess probable compatibility issues.
• 35:54 - 35:58: Try and use fluorophores with as narrow emission characteristics
• 35:58 - 36:01: as possible to minimise overlap with other fluorophores
• 36:01 - 36:03: of longer wavelength.
• 36:03 - 36:05: Stain the least abundant protein with a fluorophore
• 36:05 - 36:07: with the highest quantum yield.
• 36:07 - 36:11: If two or more proteins are expected to be of similar abundance,
• 36:11 - 36:14: try and reduce the concentration of the fluorophore
• 36:14 - 36:17: with the shortest wavelength to try and lessen the degree of bleed-through
• 36:17 - 36:19: into the ones with longer wavelength.
• 36:20 - 36:22: Balance the filter sets on the microscope carefully
• 36:22 - 36:25: to match the spectral absorption and emission profiles
• 36:25 - 36:28: of each fluorophore as closely as possible.
• 36:29 - 36:32: Balance the exposure and neutral density filter settings
• 36:32 - 36:36: on the microscope to fine-tune overly-aggressive fluorescent signals.
• 36:36 - 36:40: And finally, before conducting the actual multi-staining experiment,
• 36:40 - 36:43: observe each fluorophore separately in situ
• 36:43 - 36:46: using the filter sets of the other fluorophores being used
• 36:46 - 36:49: to practically assess any bleed-over issues.
• 36:49 - 36:52: If necessary, adjust experimental design
• 36:52 - 36:55: within parameters described above.
• 36:58 - 37:01: Although this webinar isn’t focused on multiple immunostaining
• 37:01 - 37:03: using enzyme reporter labels,
• 37:03 - 37:07: I thought it would be a good idea to briefly compare enzymatics to fluorescence.
• 37:08 - 37:10: As we have been discussing,
• 37:10 - 37:13: fluorescence has a wide range of available reporter labels
• 37:13 - 37:16: and has been long proved to be extremely sensitive,
• 37:16 - 37:19: with each label being observed in isolation with relative ease.
• 37:19 - 37:23: However, it has several disadvantages when compared to enzymatic.
• 37:23 - 37:26: Firstly, when in close proximity to each other,
• 37:26 - 37:31: fluorochrome signals can be quenched due to the transfer of energy
• 37:31 - 37:34: from an excited fluorochrome to another.
• 37:34 - 37:37: Secondly, fading or photobleaching of the fluorescent signal
• 37:37 - 37:40: can occur during storage or analysis.
• 37:40 - 37:44: Lastly, specimen auto-fluorescence is exacerbated by aldehyde fixation,
• 37:44 - 37:47: potentially interfering with results interpretation.
• 37:47 - 37:50: Enzyme and chromogen combinations
• 37:50 - 37:53: allow for two or more end-product precipitate colors to be used,
• 37:53 - 37:56: negating the problems associated with fluorescence.
• 37:56 - 37:59: And finally, when in close proximity to each other,
• 37:59 - 38:02: fluorochrome signals can be quenched due to the transfer of energy
• 38:02 - 38:05: negating the problems associated with fluorescence.
• 38:05 - 38:08: Also, since the colored precipitate stays localized on the tissue
• 38:08 - 38:11: independently of the detection system,
• 38:11 - 38:14: this allows for the elution of primary and detection system antibodies
• 38:14 - 38:17: by heat-induced epitope retrieval,
• 38:17 - 38:20: allowing subsequent rounds of immunostaining to occur
• 38:20 - 38:23: without any fears of unwanted antibody cross-reactivity.
• 38:23 - 38:27: With both fluorescence and enzyme chromogen products,
• 38:27 - 38:30: the evolution of spectral unmixing software
• 38:30 - 38:33: allows the isolation of signals,
• 38:33 - 38:36: even when co-localization is indistinguishable with the human eye,
• 38:36 - 38:39: allowing numerous different labels to be used.
• 38:44 - 38:49: Let’s now discuss an actual multiple immunostaining technique.
• 38:49 - 38:52: Simply put, a multiple staining experiment
• 38:52 - 38:57: is a combination of two or more individual single-label staining techniques.
• 38:58 - 39:01: The protocol discussed in the first half of this webinar
• 39:01 - 39:04: is a good basis for a multi-staining protocol,
• 39:04 - 39:07: inserting the necessary incubation and wash steps where appropriate.
• 39:07 - 39:11: The use of highly cross-absorbed or pre-absorbed secondary antibodies
• 39:11 - 39:16: is highly recommended in all cases to help minimize any cross-reactivity.
• 39:16 - 39:19: Optimizing the specific concentrations, etc.,
• 39:19 - 39:22: of each of the single-labeling techniques in isolation
• 39:22 - 39:25: is strongly recommended before combining them
• 39:25 - 39:27: in a multiple labeling experiment.
• 39:27 - 39:30: The main concern is how to avoid the individual components
• 39:30 - 39:34: of each of the separate detection systems from cross-reacting.
• 39:34 - 39:39: Multiple staining techniques themselves can be broken down into two groups.
• 39:39 - 39:43: Simultaneous, where the primary antibodies are applied together,
• 39:43 - 39:45: followed by the detection systems,
• 39:45 - 39:48: and sequential, where one single-label staining technique
• 39:48 - 39:51: is completed before the other.
• 39:52 - 39:56: We shall begin by taking a look at simultaneous staining.
• 39:58 - 40:01: If the species used to raise the primary antibodies
• 40:01 - 40:03: are phylogenetically different enough,
• 40:03 - 40:06: they can be applied to the specimen together.
• 40:06 - 40:09: A primary antibody combination raised in mouse and rabbit
• 40:09 - 40:12: would be considered sufficiently phylogenetically different,
• 40:12 - 40:15: whereas a combination raised in mouse and rat would not.
• 40:15 - 40:19: Secondary antibodies raised in the same species against those species
• 40:20 - 40:24: bearing different fluorophores can then be applied at the same time.
• 40:24 - 40:28: Simultaneous staining can also be achieved using primary antibodies
• 40:28 - 40:32: that are raised in species that are not sufficiently phylogenetically different,
• 40:32 - 40:34: even raised in the same species,
• 40:34 - 40:36: if the antibodies are directly labeled
• 40:36 - 40:40: with a different fluorescent label per primary antibody.
• 40:40 - 40:44: Of course, this is only viable if all of the antigens are very abundant
• 40:44 - 40:47: and a low degree of signal amplification is required.
• 40:47 - 40:50: Simultaneous staining can be performed using primary antibodies
• 40:50 - 40:54: that are raised in the same species if they are of different isotypes,
• 40:54 - 40:57: and isotype-specific secondaries are used.
• 41:01 - 41:06: As you can see, it is quite limited in what you can do in simultaneous staining,
• 41:06 - 41:09: especially in terms of signal amplification,
• 41:09 - 41:13: which is where sequential staining is more advantageous.
• 41:14 - 41:18: Sequential staining is much more flexible than simultaneous staining,
• 41:18 - 41:21: allowing additional signal amplification to be performed
• 41:21 - 41:26: rather than just secondary antibodies for use on low-abundance antigens.
• 41:26 - 41:29: The most sensitive detection system should be reserved for the antigen
• 41:29 - 41:32: that is expected to be the least abundant.
• 41:32 - 41:36: Particular attention needs to be paid to the order that the reagents are added.
• 41:36 - 41:40: An example would be three primary antibodies, all raised in mouse,
• 41:40 - 41:43: one directly conjugated to a fluorophore,
• 41:43 - 41:47: one directly conjugated to biotin, and one unconjugated.
• 41:47 - 41:52: Step one, incubate the specimen with the unconjugated mouse primary.
• 41:52 - 41:56: Step two would be to incubate the specimen with a fluorophore-labeled secondary,
• 41:56 - 41:59: such as goat anti-mouse.
• 41:59 - 42:02: Next, incubate the specimen with mouse serum
• 42:02 - 42:06: to ensure that the goat anti-mouse antibodies are saturated.
• 42:06 - 42:10: Step four is to incubate the specimen with the biotinylated mouse primary.
• 42:10 - 42:13: In step five, we would then incubate the specimen
• 42:13 - 42:16: with a fluorophore-labeled ABC complex.
• 42:16 - 42:19: And finally, step six is to incubate the specimen
• 42:19 - 42:23: with the directly fluorophore-conjugated mouse antibody.
• 42:23 - 42:25: This would result in three-color staining
• 42:25 - 42:28: with no potential for the reagents to cross-react.
• 42:28 - 42:31: Please also note that there would be buffer washes
• 42:31 - 42:34: in between each step in the sequence above.
• 42:35 - 42:39: Another method when using antibodies raised in the same species
• 42:39 - 42:43: is to technically change the species of one of the primary antibodies
• 42:43 - 42:46: by adding a bridging antibody of some description.
• 42:46 - 42:49: An example would be two primary antibodies,
• 42:49 - 42:52: both raised in mouse, one unconjugated,
• 42:52 - 42:55: and one directly conjugated to HRP.
• 42:55 - 43:04: Step one, incubate the specimen with the first unconjugated mouse primary.
• 43:04 - 43:06: Step two would be to incubate the specimen
• 43:06 - 43:10: with a fluorophore-labeled secondary, such as goat anti-mouse.
• 43:10 - 43:12: Next, incubate the specimen with mouse serum
• 43:12 - 43:16: to ensure that the goat anti-mouse antibodies are saturated.
• 43:16 - 43:18: Step four is then to incubate the specimen
• 43:18 - 43:22: with the second HRP-conjugated mouse primary.
• 43:22 - 43:25: In step five, we would then incubate the specimen
• 43:25 - 43:29: with an anti-HRP antibody, such as a rabbit anti-HRP.
• 43:29 - 43:32: And finally, step six is to incubate the specimen
• 43:32 - 43:36: with a fluorophore-conjugated goat anti-rabbit antibody.
• 43:36 - 43:39: Alternatively, the second mouse antibody
• 43:39 - 43:43: could have been directly biotinylated and an ABC system used.
• 43:46 - 43:49: Again, please also note that there would be buffer washes
• 43:49 - 43:52: in between each step in the sequence above.
• 43:53 - 43:57: Lastly, let us consider the controls that must be employed
• 43:57 - 43:59: when doing multiple immunostaining.
• 44:02 - 44:04: As with all immunostaining techniques,
• 44:04 - 44:08: controls are essential to validate that any staining is a true result
• 44:08 - 44:12: and not from an intrinsic property of the detection system or specimen.
• 44:12 - 44:14: Since multiple staining is a combination
• 44:14 - 44:18: of two or more individual single-label staining techniques,
• 44:18 - 44:20: the number of individual reagent controls
• 44:20 - 44:24: that could be used for each detection system can be rather daunting,
• 44:24 - 44:27: especially given the time constraints of the modern laboratory.
• 44:27 - 44:30: At a minimum, the following should be done.
• 44:30 - 44:33: Firstly, positive controls,
• 44:33 - 44:35: performing the separate single-staining techniques
• 44:35 - 44:39: for each primary antibody and detection system combination.
• 44:39 - 44:42: And lastly, negative reagent controls,
• 44:42 - 44:44: performing separate single-staining techniques
• 44:44 - 44:48: for each detection system minus the primary antibodies.
• 44:49 - 44:53: OK, so my part of this webinar is almost over,
• 44:53 - 44:56: but with regards to multiple immunostaining,
• 44:56 - 44:59: if you remember nothing else, remember the following.
• 44:59 - 45:02: Numerous staining combinations are possible
• 45:02 - 45:05: as long as careful attention is paid to the experimental design.
• 45:05 - 45:09: Select fluorophores so that their emission spectra do not overlap
• 45:09 - 45:12: and match them to the filters on your microscope.
• 45:12 - 45:15: Carefully consider the various detection systems available,
• 45:15 - 45:17: the potential cross-reactivities,
• 45:17 - 45:19: and the order that the reagents are added.
• 45:19 - 45:22: Finally, always use the appropriate controls
• 45:22 - 45:25: and perform each separate staining technique in isolation
• 45:25 - 45:28: in order to optimise the concentration of each reagent
• 45:28 - 45:32: and to assess potential bleed-through.
• 45:32 - 45:34: Thank you very much for your time,
• 45:34 - 45:36: and I hope that you found it useful.
• 45:36 - 45:39: I’ll be back in a few minutes to answer some of your questions,
• 45:39 - 45:42: but in the meantime, over to you, Judith.
• 45:45 - 45:49: Thank you, Simon, for such a detailed seminar, and hello again.
• 45:49 - 45:52: I would like to take this opportunity to tell you a bit more
• 45:52 - 45:56: about Abcam’s IHC-ICC resources and products
• 45:56 - 46:01: that will help you to improve your cell imaging experiments.
• 46:01 - 46:06: Rabbit monoclonals, or RabMAbs, offer high affinity and specificity,
• 46:06 - 46:12: which results in high sensitivity and low background staining.
• 46:12 - 46:18: makes them ideal reagents for use in demanding applications
• 46:18 - 46:24: such as IHC on formalin-fixed or paraffin-embedded tissues.
• 46:24 - 46:28: The best experience is when RabMAbs has been tested
• 46:28 - 46:31: on multiple human tissue arrays for IHC
• 46:31 - 46:36: or on multiple samples for ICC-IS.
• 46:36 - 46:39: RabMAbs also offer diverse epitope recognition
• 46:39 - 46:43: of human protein targets and their mouse orthologs,
• 46:43 - 46:48: so there is no need to generate a separate surrogate antibody.
• 46:48 - 46:50: Due to the fact that they are rabbit-generated,
• 46:50 - 46:55: they are ideal for use on mouse or rat tissue samples.
• 46:55 - 46:59: It can also be easily paired with mouse or rat monoclonal antibodies
• 46:59 - 47:01: for simultaneous staining.
• 47:01 - 47:07: For further information, please visit abcam.com RabMAb.
• 47:07 - 47:09: For staining with your RabMAbs,
• 47:09 - 47:13: we recommend Alexa Fluor conjugated secondary antibodies.
• 47:13 - 47:22: These antibodies are available conjugated to Alexa Fluor 488, 555, 594, and 647.
• 47:22 - 47:26: All of these secondary antibodies have been extensively tested
• 47:26 - 47:31: in the Abcam laboratories to guarantee bright staining and low background.
• 47:31 - 47:34: The selection of preabsorbed antibodies is large
• 47:34 - 47:38: to ensure low species cross-reactivity.
• 47:38 - 47:43: The dilution range for all products is between 1 in 200 until 1 in 1,000,
• 47:43 - 47:47: giving you roughly 250 stainings.
• 47:47 - 47:54: In addition to that, all products are competitively priced.
• 47:54 - 48:01: For more information, please go to abcam.com Alexa.
• 48:01 - 48:06: Abcam’s catalog includes a whole range of cell imaging tools for multicolor staining.
• 48:06 - 48:11: Discover our Cytopainter range of kits for staining of actin filaments,
• 48:11 - 48:15: mitochondria, and lysosomes in multiple colors.
• 48:15 - 48:18: It is an easy way to study co-localization
• 48:18 - 48:22: without having to fiddle around with multiple antibodies.
• 48:22 - 48:29: Cytopainter kits can be used in combination with secondary antibodies and nuclear dyes.
• 48:29 - 48:37: An example of this is the IHC image of mouse embryo bodies shown on the top right corner.
• 48:37 - 48:40: For trouble-free staining of nuclei,
• 48:40 - 48:45: why not try far-red dyes that will display nuclear staining in just five minutes?
• 48:45 - 48:49: Included in this range are DRUG-5 and DRUG-7,
• 48:49 - 48:53: which can be used for staining live or fixed cells.
• 48:53 - 48:57: The bottom image shows nuclei stained with DRUG-7.
• 48:57 - 49:02: The blue staining originates from an AMCA-conjugated secondary antibody.
• 49:02 - 49:09: We have an extensive portfolio of validated secondary antibodies for cell imaging,
• 49:09 - 49:17: including our preabsorbed Alexa and DyLight-conjugated secondary antibodies for minimal cross-reactivity.
• 49:17 - 49:23: Our range also includes Chromo-conjugated secondary antibodies for STED microscopy
• 49:23 - 49:29: and AbGold-conjugated secondary antibodies for high-resolution electron microscopy.
• 49:29 - 49:34: In order to increase tissue penetration in your IHC experiments,
• 49:34 - 49:38: we recommend you try one of our PAP2 fragment antibodies.
• 49:38 - 49:43: For non-fluorescent imaging or enzymatic detection,
• 49:43 - 49:48: Abcam also offers a comprehensive range of products for IHC.
• 49:48 - 49:52:  Included in the portfolio are exposed IHC kits,
• 49:52 - 49:58:  which provide greater sensitivity in comparison to polymers and ABCs detection systems
• 49:58 – 50:03:  This is achieved through a smaller detection complex.
• 50:04 - 50:09:  Our IHC portfolio also includes the classical biotin and streptavidin kits,
• 50:09 - 50:11:  in addition to various reagents.
• 50:12 - 50:15:  If you would like to know more about our cell imaging products,
• 50:15 - 50:18:  please visit abcam.com/imaging.
• 50:18 - 50:24:  On this page, you can find more detailed information and can zoom in on some of our imges.
• 50:26 - 50:31:  A complication often experienced when using mouse antibodies on mouse tissue
• 50:31 - 50:34:  is a high level of background.
• 50:34 - 50:30:  This is due to the secondary antibody binding endogenous mouse IgG.
• 50:32 - 50:46:  A product we offer that provides a solution to this is the mouse-on-mouse IHC detection kit.
• 50:48 - 50:52:  This polymer-based detection kit contains a blocking reagent
• 50:52 - 50:54:  to block endogenous mouse IgGs,
• 50:54 - 50:59:  ensuring minimal background in addition to a simple and reliable protocol.
• 51:00 - 51:04:  More about this product can be found at abcam.com/mon.
• 51:07 - 51:09:  As Simon mentioned during the webinar,
• 51:09 - 51:11:  it is important to prevent photobleaching.
• 51:12 - 51:16:  For that purpose, Abcam offers a range of fluoroshield mounting reagents.
• 51:17 - 51:20:  These are available with or without nuclear dyes,
• 51:20 - 51:22:  such as DAPI and propidium iodide.
• 51:25 - 51:30:  Abcam's scientific support team is here to answer any questions you may have.
• 51:31 - 51:35:  The team members are multilingual and offer support in a range of languages,
• 51:35 - 51:40:  including French, Spanish, German, Chinese, and Japanese.
• 51:41 - 51:45:  You can contact them in the US, UK, Hong Kong, and Japan.
• 51:47 - 51:51:  I would like to finish by thanking Simon and all of you for attending
• 51:52 - 51:57:  and would like to wish you all the best for your future IHC-ICC experiments.
• 52:00 - 52:01:  Thank you, Judith.
• 52:02 - 52:05:  As with the first webinar, we've had some really excellent questions coming through.
• 52:06 - 52:09:  Due to time constraints, I can only go through about three of them,
• 52:09 - 52:11:  so I've picked out three really good ones.
• 52:12 - 52:13:  Kevin has asked,
• 52:13 - 52:16:  I have high background in my multi-labeling experiments,
• 52:16 - 52:17:  why could that be?
• 52:18 - 52:20:  It could be due to so many reasons, Kevin,
• 52:21 - 52:23:  such as over-retrieving antigens,
• 52:23 - 52:26:  maybe try less retrieval time, a different buffer or enzyme,
• 52:26 - 52:29:  insufficient blocking, too short a blocking time,
• 52:29 - 52:30:  or the wrong serum is used.
• 52:31 - 52:33:  Your tissues could be rich in endogenous enzymes,
• 52:33 - 52:35:  or they're not sufficiently quenched.
• 52:36 - 52:38:  Tissue could also be rich in endogenous biotin,
• 52:38 - 52:41:  if used in an ABC detection system, or what else.
• 52:42 - 52:43:  Species cross-reactivity,
• 52:44 - 52:46:  so the detection reagents could be cross-reacting,
• 52:46 - 52:49:  or the secondary could simply be binding to immunoglobulins in the blocking serum.
• 52:50 - 52:53:  Maybe you want to try using pre-absorbed secondaries for additional specificity.
• 52:53 - 52:57:  The antibodies that you're using may be unsuitable for IHC or IF,
• 52:58 - 53:00:  or may also generally just be nonspecific,
• 53:01 - 53:04:  and there could also be high levels of water fluorescence in your specimen.
• 53:04 - 53:06:  I don't know your exact experimental design.
• 53:07 - 53:09:  There are numerous other possibilities,
• 53:09 - 53:11:  too far away to cover right now,
• 53:12 - 53:14:  but if I were you, I'd go through all your protocol line by line,
• 53:14 - 53:17:  and see if you can identify any of the above possibilities.
• 53:17 - 53:19:  Don't forget to include all of the necessary controls
• 53:19 - 53:21:  for each of the single staining procedures,
• 53:21 - 53:23:  and perform each of them in their entirety in isolation.
• 53:26 - 53:31:  That may hold the key to where your background staining is originating.
• 53:31 - 53:33:  Anyway, good luck with that.
• 53:33 - 53:41:  Okay, so another question. Mandit says, I am using donkey serum for blocking and anti-sheep secondary.
• 53:41 - 53:44:  My background is really high. What can you suggest?
• 53:44 - 53:52:  Well, the most obvious thing that springs to mind is that even though your secondary antibody is raised against sheep immunoglobulins,
• 53:52 - 53:54:  it's also detecting the donkey to some degree.
• 53:54 - 54:00:  It's all to do with phylogeny so donkey and sheep are both ungulates, correct me if I'm wrong.
• 54:00 - 54:03:  And for that reason, the amino acid sequence
• 54:03 - 54:06:  in their immunoglobulin heavy chains will be quite similar,
• 54:06 - 54:08:  hence the possibility for cross-reactivity.
• 54:08 - 54:16:  I guess there are several things that you can do. Number one, you can use an anti-sheep that's been pre-absorbed against donkey, and see if that makes a difference.
• 54:16 - 54:19:  Or you can not use donkey serum at all for blocking.
• 54:19 - 54:22:  Omit it completely, see if the problem stops. Or I guess as well,
• 54:22 - 54:24:  you can see if you can find a primary antibody towards your antigen
• 54:25 - 54:26:  that is not raised in sheep,
• 54:27 - 54:28:  so you can use a different secondary.
• 54:29 - 54:31:  Try and find a mouse or rabbit primary, for instance,
• 54:32 - 54:34:  and use a secondary that is raised in goat, for example.
• 54:35 - 54:37:  Give those a go and see if they help.
• 54:38 - 54:39:  Okay, we've just got time for one more.
• 54:40 - 54:41:  The last one's from Stephen.
• 54:42 - 54:43:  He'd like to know,
• 54:44 - 54:45:  you mentioned spectral mixing software.
• 54:45 - 54:47:  Could you tell us a bit more about this?
• 54:48 - 54:50:  I'm going to be completely honest, Stephen.
• 54:51 - 54:52:  I haven't had much experience of actually using it myself,
• 54:53 - 54:54:  but I've attended several lectures
• 54:55 - 54:56:  where people have been very impressed with it
• 54:57 - 54:58:  and have shown some of the good results
• 54:59 - 55:00:  that they've obtained with it in action.
• 55:01 - 55:02:  And believe me, if I controlled the department's budget,
• 55:03 - 55:04:  I'd buy one tomorrow.
• 55:05 - 55:06:  So in a nutshell, it's microscopy software
• 55:07 - 55:09:  that is much more sensitive to color than the human eye.
• 55:10 - 55:11:  In other words, it can distinguish
• 55:12 - 55:13:  between very subtle color differences in labels,
• 55:14 - 55:17:  especially when they're merged in co-localization experiments,
• 55:18 - 55:19:  allowing much easier separation
• 55:20 - 55:21:  and visualization of the signals.
• 55:22 - 55:24:  This, therefore, lets you use reporter labels
• 55:25 - 55:26:  that spectrally overlap both chromogenic
• 55:27 - 55:29:  and end-products and fluorescent labels,
• 55:30 - 55:31:  allowing for additional labels to be used
• 55:32 - 55:33:  in a multi-labeling experiment.
• 55:34 - 55:35:  Otherwise, you just wouldn't have been able
• 55:36 - 55:37:  to distinguish a part with the human eye.
• 55:38 - 55:40:  It's well worth the money if you've got the money to spare.