Step-by-step IHC demonstration: Immunohistochemistry protocol and staining techniques

Video transcript

• 00:07 - 00:11: Hello everyone, we know that immunohistochemistry or IHC
• 00:11 - 00:17: is a method that uses antigen-antibody interactions to show the distribution and
• 00:17 - 00:20: location of antigens in tissue sections.
• 00:21 - 00:27: IHC is often used to diagnose abnormal cells and tissues in diseases such as
• 00:27 - 00:28: cancer.
• 00:29 - 00:35: IHC can be used to define normal cells and disease processes and can help
• 00:35 - 00:39: discover potential methods for disease treatment.
• 00:40 - 00:45: IHC standing relies on antibodies that recognize target proteins.
• 00:47 - 00:52: The antigen antibody interaction is mainly visualized through 2 detection
• 00:52 - 00:53: methods.
• 00:54 - 01:00: One, is the chromogenic method, in which an enzyme-conjugated antibody catalyzes the
• 01:00 - 01:03: substrate to produce a colored precipitation.
• 01:04 - 01:10: The other is the fluorescence detection method, where a fluorophore conjugated
• 01:10 - 01:13: antibody produces fluorescent signals
• 01:14 - 01:18: In actual IHC experiments, due to incorrect or suboptimal
• 01:18 - 01:23: experimental conditions, staining artifacts like high background,
• 01:23 - 01:27: No staining and nonspecific staining may occur.
• 01:28 - 01:32: Next, we will demonstrate the experimental steps of IHC.
• 01:33 - 01:40: We will mainly cover sample preparation, antigen retrieval,
• 01:40 - 01:45: blocking antigen incubation and detection.
• 01:47 - 01:50: First, we need to prepare tissue samples for
• 01:50 - 01:51: immunohistochemistry.
• 01:52 - 01:57: Here we take formalin-fixed, paraffin-embedded or FFP tissue samples
• 01:57 - 01:59: for illustration.
• 02:00 - 02:05: After collecting the tissue sample, it needs to be fixed and dehydrated, and
• 02:05 - 02:06: finally embedded in paraffin.
• 02:07 - 02:14: The paraffin-embedded tissue is then sectioned. Fresh tissue for IHC detection
• 02:14 - 02:21: requires fixation to prevent autolysis and degradation of the ex vivo tissue, and to
• 02:21 - 02:24: preserve the antigenicity of the proteins.
• 02:25 - 02:30: Common fixatives include cross-linking reagents such as aldehyde,
• 02:30 - 02:35: fixatives like formalin, and organic solvents such as alcohol and
• 02:35 - 02:36: acetone.
• 02:37 - 02:42: The choice of fixative depends on the protein to be detected and the detection
• 02:42 - 02:43: method.
• 02:44 - 02:50: The main fixation methods include perfusion fixation, emergent fixation,
• 02:50 - 02:52: and freeze fixation.
• 02:53 - 02:57: In this illustration, we use perfusion-fixed mouse brain tissue
• 02:57 - 02:59: and further fix it with emergent fixation.
• 03:02 - 03:05: Cut the tissue block to an appropriate size,
• 03:05 - 03:10: handle it carefully during sampling to avoid mechanical damage,
• 03:10 - 03:14: and fix the sample as soon as possible after removal.
• 03:21 - 03:28: Immerse the processed tissue block in the fixative for 18 to 24 hours.
• 03:28 - 03:34: The fixation time need to be optimized according to the size and type of the
• 03:34 - 03:35: tissue block.
• 03:36 - 03:42: Insufficient fixation may lead to edge standing and over fixation may reduce
• 03:42 - 03:43: protein signals.
• 03:44 - 03:50: The fixed tissue must undergo the hydration and clearing before paraffin
• 03:50 - 03:55: impacted as water and paraffin are not miserable, and the hydration makes it
• 03:55 - 03:59: easier for paraffin to infiltrate the tissue in film hold.
• 04:00 - 04:05: Immerse the fixed tissue in ethanol solutions of different concentrations for
• 04:05 - 04:06: suitable times.
• 04:07 - 04:12: This procedure uses 75% ethanol for 30 minutes,
• 04:12 - 04:18: 85% ethanol twice each for 30 minutes,
• 95% ethanol twice,
• 04:18 - 04:24: the first for 30 minutes and the second overnight,
• 04:24 - 04:30: and anhydrous ethanol twice each for 30 minutes.
• 04:30 - 04:39: Then immerse in xylene solution twice each for 15 minutes to complete the
• 04:39 - 04:43: dehydration and clearing steps.
• 04:51 - 04:55: After dehydration and clearing, this can be embedded in paraffin.
• 04:56 - 05:00: First, place the xylene in the tissue with paraffin.
• 05:00 - 05:07: Transfer the tissue from the xylene solution to melted paraffin and immerse
• 05:07 - 05:09: it three times each for 30 minutes.
• 05:12 - 05:13: Then proceed.
• 05:13 - 05:15: Proceed to embedding.
• 05:15 - 05:20: Set the parameters of the paraffin embedding machine in advance,
• 05:20 - 05:25: then add melted paraffin into the preheated embedded mold to about 2/3 of
• 05:25 - 05:26: its volume.
• 05:26 - 05:31: Use preheated tweezers to place the tissue in the embedded mold and adjust
• 05:31 - 05:33: its position appropriately.
• 05:34 - 05:41: Place the embedded cassette on top and continue adding paraffin until the
• 05:41 - 05:47: embedded cassette's lower edge is reached, avoiding bubbling formation.
• 05:52 - 05:57: After embedding, move the paraffin block to a cold plate
• 05:57 - 06:01: and cool it for an appropriate time or overnight.
• 06:01 - 06:04: In this illustration, we let it cool overnight.
• 06:15 - 06:20: Remove the cooled paraffin block from the embedding mold,
• 06:20 - 06:23: a process known as demolding.
• 06:25 - 06:32: Note that paraffin blocks cooled to room temperature need to be further cooled
• 06:32 - 06:37: down with a -20°C tweezer for an appropriate time,
• 06:37 - 06:40: like 10 minutes before demolding.
• 06:40 - 06:44: The paraffin block can be stored at room temperature for a long time or
• 06:44 - 06:47: proceed to the next step of sectioning.
• 06:48 - 06:50: The embedded tissue now can be sectioned.
• 06:51 - 06:54: Cooling the paraffin blocks makes sectioning easier.
• 06:54 - 06:58: It's recommended to place the paraffin block on ice or in a
• 06:58 - 06:59: -20°C freezer before sectioning.
• 07:01 - 07:03: Start the tissue section warmer.
• 07:03 - 07:07: Set in a temperature according to the tissue characteristics.
• 07:07 - 07:10: This procedure sets it at 42°C.
• 07:12 - 07:19: Install and adjust the blade parameters according to the microtome manufacturer's
• 07:19 - 07:20: recommendations.
• 07:20 - 07:24: Fixed and positioned the paraffin embedded tissue block.
• 07:24 - 07:29: Then slowly move the blade towards the paraffin block to trim it.
• 07:30 - 07:36: Test Cut a few thin sections to confirm the appropriate position of the paraffin
• 07:36 - 07:37: block.
• 07:40 - 07:44: The well-trimmed tissue block should reveal the tissue surface.
• 07:45 - 07:47: The trimming thickness is about 30 micrometers.
• 07:49 - 07:52: After trimming, set the tissue thickness to about 3
• 07:52 - 07:53: micrometers.
• 07:53 - 07:57: Transfer suitable sections to the tissue section warmer.
• 07:57 - 08:02: Use tweezers to gently tap the sections to separate them.
• 08:05 - 08:11: Once the tissue sections are flat and wrinkles disappear, they can be picked up.
• 08:17 - 08:22: Use pre-coated slides labeled with sample information to pick up the sections from
• 08:22 - 08:26: tissue section warmer and place them on the drying rack.
• 08:27 - 08:32: Be careful to avoid bubbles between the sections and the slides.
• 08:33 - 08:37: The joint step should be at an appropriate temperature and time.
• 08:38 - 08:42: This example uses 62°C for two hours.
• 08:42 - 08:47: For delicate or central nervous system tissues,
• 08:47 - 08:53: a lower drying temperature is recommended, such as 37°C for 24 hours.
• 08:59 - 09:01: Before starting IHC staining.
• 09:01 - 09:05: The T sections need to be deparaffinized and rehydrated.
• 09:06 - 09:12: Incomplete dehydration makes standing difficult in a film hood.
• 09:12 - 09:18: Immerse the sections in xylene twice each for 15 minutes,
• 09:18 - 09:24: then rehydrate them sequentially with anhydrous ethanol.
• 9:35 - 09:52: 95% ethanol, 85% ethanol, and 75% ethanol solutions each for 5
• 09:52 - 09:59: minutes, repeated twice.
• 10:03 - 10:10: Then wash the sections in the ionized water on a shaker three times each for 5
• 10:10 - 10:11: minutes.
• 10:16 - 10:20: Since aldehyde fixatives are used in this procedure,
• 10:20 - 10:23: the paraffinized sections need antigen retrieval.
• 10:24 - 10:29: Then block the background and incubate with primary and secondary antibodies
• 10:29 - 10:31: before proceeding to the next step.
• 10:34 - 10:41: Most aldehyde-fixed samples for methylene bridges that cross-link proteins during
• 10:41 - 10:41: fixation.
• 10:42 - 10:45: Antigen retrieval can break these cross-links,
• 10:45 - 10:48: exposing antigen sites for antibody binding.
• 10:49 - 10:55: Antigen retrieval methods mainly include heat-induced antigen retrieval and enzyme
• 10:55 - 10:57: induced antigen retrieval.
• 10:58 - 11:03: Choice depends on the antigen type, tissue properties, and fixation method.
• 11:03 - 11:10: This example uses Abcam PH9 EDTA antigen retrieval buffer for heat-induced antigen
• 11:10 - 11:11: retrieval.
• 11:13 - 11:22: Immerse the sections in antigen retrieval buffer and then place them in an antigen
• 11:22 - 11:24: retrieval cooker.
• 11:29 - 11:36: This procedure sets it at 110°C for 15 minutes.
• 11:41 - 11:44: After the heat-induced engine retrieval program,
• 11:44 - 11:49: remove the sections and let them cool to room temperature naturally.
• 11:50 - 11:57: Then wash the sections in the deionized water on the shaker three times each for
• 11:57 - 12:02: 5 minutes to thoroughly remove the antigen retrieval buffer.
• 12:05 - 12:11: After the antigen retrieval, we will proceed to block the background
• 12:11 - 12:14: proteins using 10% gold serum.
• 12:16 - 12:20: Depending on the type of IHC tissue and reagents,
• 12:20 - 12:25: additional blocking, such as peroxidase blocking, aldehyde blocking,
• 12:25 - 12:30: and alkaline phosphatase blocking may be required.
• 12:30 - 12:35: This procedure uses HRP-labeled secondary antibodies,
• 12:35 - 12:42: So treat sections with 3% hydrogen peroxide for 10 minutes to block
• 12:42 - 12:44: endogenous peroxidase.
• 12:50 - 12:56: Then wash the sections in the ionized water three times each for 5 minutes to
• 12:56 - 12:59: remove excess hydrogen peroxide.
• 13:13 - 13:22: After removing excess deionized water, use an IHCP pen to draw a hydrophobic
• 13:22 - 13:26: barrier around the tissue sections.
• 13:27 - 13:34: Soak the sections in 1X TBST, then add an appropriate amount of 10%
• 13:34 - 13:38: gold serum blocking solution to the sections.
• 13:46 - 13:51: The blocking time should be adjusted according to the blocking agent type.
• 13:52 - 14:01: This procedure blocks at room temperature in a humidified chamber for 0.5 hours.
• 14:05 - 14:12: Then wash the sections in 1X TBST solution on a shaker three times each for
• 14:12 - 14:16: 5 minutes to remove excess blocking solution.
• 14:31 - 14:37: After antigen retrieval and blocking, the sections need to be incubated with
• 14:37 - 14:38: the primary antibody.
• 14:39 - 14:44: Add an appropriate amount of primary antibody to the antibody diluent and mix
• 14:44 - 14:45: well.
• 14:46 - 14:50: Dilute the primary antibody according to the recommended concentration in the
• 14:50 - 14:51: antibody data sheet.
• 14:52 - 14:59: For the first use of an antibody, it is recommended to perform a gradient
• 14:59 - 15:05: dilution to determine the appropriate optimal working concentration.
• 15:08 - 15:15: Add the diluted primary antibody working solution to the sections and place them
• 15:15 - 15:17: in a humidified chamber.
• 15:37 - 15:48: After adding the primary antibody to the sections and put them in a humidified
• 15:48 - 15:58: chamber incubated overnight at 4°C, before starting the experiment.
• 15:58 - 16:02: Next day, take out the humidified chamber with the
• 16:02 - 16:07: sections and let it equilibrate to the room temperature for at least half an
• 16:07 - 16:07: hour.
• 16:09 - 16:15: After primary incubation, wash the sections in 1X TBST solution
• 16:15 - 16:21: on a shaker three times each for 5 minutes to remove unbound primary
• 16:21 - 16:22: antibody.
• 16:29 - 16:35: Now, after primary antibody incubation, we will incubate the sections with
• 16:35 - 16:41: secondary antibodies conjugated with a reporter label to detect the antigen.
• 16:43 - 16:49: Secondary antibodies are usually selected based on the species of the primary
• 16:49 - 16:50: antibody.
• 16:51 - 16:55: To obtain stronger signals, it is recommended to use HRP polymer
• 16:55 - 16:57: conjugated secondary antibodies.
• 16:59 - 17:04: Add an appropriate amount of ready-to-use secondary antibody to the sections and
• 17:04 - 17:09: adjust the amount and incubation time according to the type of the secondary
• 17:09 - 17:10: antibody.
• 17:10 - 17:15: This procedure incubates at room temperature for 0.5 hours.
• 17:16 - 17:19: If using fluorophore-conjugated secondary antibodies,
• 17:19 - 17:22: you need to incubate it in the dark.
• 17:24 - 17:30: After secondary antibody incubation, wash the sections thoroughly in 1X TBST
• 17:30 - 17:35: solution on a shaker three times each for
• 5 minutes to remove nonspecific binding.
• 17:36 - 17:41: After incubation with antibodies, sections can be detected using
• 17:41 - 17:45: chromogenic or fluorescence detection methods.
• 17:46 - 17:50: This procedure uses HRP labeled secondary antibodies
• 17:50 - 17:56: so enzyme substrate thinning is performed, that is, chromogenic detection.
• 17:58 - 18:03: For DAB chromogenic detection, add an appropriate amount of DAB
• 18:03 - 18:05: substrate to the sections.
• 18:26 - 18:33: After adding the DAB substrate, incubate at room temperature for five to
• 18:33 - 18:36: 10 minutes until the color develops.
• 18:37 - 18:41: Monitoring the standing process and the color development.
• 18:41 - 18:48: Stop the reaction by washing the sections in the ionized water on the shaker for three
• 18:48 - 18:53: times each for 5 minutes to remove excess DAB solution.
• 19:04 - 19:10: After color development, perform hematoxylin counterstaining to
• 19:10 - 19:13: visualize the tissue structure.
• 19:16 - 19:21: Immerse the sections in hematoxylin standing solution for one to two minutes.
• 19:28 - 19:32: Wash the sections in running tap water to remove excess hematoxylin.
• 19:41 - 19:47: Differentiate the sections in 1% acid alcohol for a few minutes if needed to
• 19:47 - 19:49: adjust the standing intensity.
• 19:50 - 19:53: Then wash the sections in running tap water.
• 19:53 - 19:59: Then, immerse in 1% ammonium hydroxide solution for one minute to turn the
• 19:59 - 20:06: color blue and wash the sections in running tap water for at least 10 minutes
• 20:08 - 20:12: After counterstaining, sections need to be dehydrated,
• 20:12 - 20:15: cleared and mounted for long-term preservation.
• 20:16 - 20:21: In the film hood, immerse the sections in 75% ethanol.
• 20:22 - 20:30: 85% ethanol, 95% ethanol and 100% anhydrous ethanol
• 20:30 - 20:38: solutions sequentially for 5 minutes each to dehydrate,
• 20:38 - 20:50: Then, immerse the xylene solution twice each for 5 minutes to clear the sections.
• 21:04 - 21:10: Add an appropriate amount of mounting medium to the sections and cover with the
• 21:10 - 21:10: cover slip.
• 21:11 - 21:14: Avoid bubbles when mounting the cover slip.
• 21:16 - 21:20: Let the sections dry completely in a fume hood.
• 21:25 - 21:28: After drying, the sections can be stored long-term at
• 21:28 - 21:31: room temperature and observed under a microscope.
• 21:33 - 21:39: The key to obtaining high-quality IHC results is to follow the standard
• 21:39 - 21:44: procedures and optimize conditions for each step according to the sample and
• 21:44 - 21:46: antigen characteristics.
• 21:46 - 21:51: Careful handling during sectioning, subverted paraffinization,
• 21:51 - 21:57: appropriate antigen retrieval and accurate antibody incubation are
• 21:57 - 21:59: critical for successful staining.
• 22:03 - 22:08: This completes the IHC experiment procedure demonstration.
• 22:08 - 22:09: Thank you for watching.