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Get all the protocols you need for your fluorescence staining experiments in one place.
Our fluorescent ICC/IF and IHC protocols cover all aspects of your experiment, from sample preparation to immunostaining and mounting. Whether you want a protocol to follow in the lab during your experiment, or you want to understand in more detail what happens at each step, you can find the information below.
Let us take you through a quick overview into the steps of fluorescent staining as well as considerations when staining more than one antigen.
This protocol provides you with the steps to follow for cell and tissue preparation, fixation, permeabilization and fluorescent immunostaining. For the best results, we have also provided our optimized protocols, including recommended dilutions on the datasheet of our antibodies.
Fluorescent staining of tissue samples has the ability to provide higher resolution images and a greater potential to multiplex compared with chromogenic immunodetection. This comprehensive guide provides you with detailed information about each step for immunofluorescent staining of tissue samples.
Video transcript for "ICC overview"
So to take you through the procedure briefly, first of all you would need to fix the cells, you may then need to permeabilize your cells for cytoplasmic or nuclear proteins. You’ll need a blocking step, a primary antibody incubation, a secondary antibody incubation and then of course you’ll need to mount and store your slides and you may like to nuclear counterstain.
So let’s begin with fixation. Fixation is required to preserve the cells to preserve their morphology and their composition and to keep the target in a good condition and correct localisation. There’s two main ways in which you can do this; you can do paraformaldehyde crosslinking fixation or you can use organic solvents. For PFA fixation, we would recommend to fix in 4% PFA for 10 minutes only. This is quite a short fixation time but there may still be some crosslinking of proteins so it’s not suitable for all epitopes. For organic solvents such as acetone, methanol and ethanol, you need to use these at ice cold temperature, again for 5 to 10 minutes only. And you need to consider that they can affect the structure of proteins and affect the epitope of the antibody. And they will also permeabilize the cell, so we wouldn’t recommend them for membrane protein detection in normal circumstances.
So once you’ve fixed the cells you may want to store them for a while before you continue with the staining. So, in order to do this, you’ll need to dry the coverslips and the cells completely. Place them in the slide box or wrap them gently in foil so they’re protected from the light, keep them at 4°C. We would recommend to keep them up to 2 to 4 weeks, not really any longer than this before staining. And then when you’re ready to stain, you can rehydrate the samples in PBS before proceeding with the protocol, and for that you’d need to start with the blocking step.
So the blocking step is there in order to minimize the background staining from any protein antibody interactions and these are not specific to your antigen-antibody binding. So any non-specific binding you’re trying to eliminate. There are various blocking agents you can use. There’s serum, BSA, milk, casein, gelatin. There are some others as well. And we would recommend to use 5 to 10% blocking agent. For ICC, it’s usually serum or BSA that’s used. And we would recommend to use the same blocking agent throughout the entire experiment and not to change between them. You can dilute the blocking agent in PBS or TBS, and blocking can be from 20 minutes to 1 hour, and again you may find that requires some optimisation. After this step, you can wash the cells three times for 5 minutes in PBS containing 0.2% Tween detergent.
Next if you require it, you will need to do some permeabilisation. Permeabilisation is only needed for cytoplasmic and nuclear proteins. Sometimes you may need to consider it for membrane proteins that have an epitope on the cytoplasmic side of the membrane. And if you need more information you can contact our scientific support team, about the particular antibody you are using. There are a couple of methods for permeabilisation. You can fix in organic solvent and this will permeabilize the cells, or you can use 0.1 to 0.2% Triton detergent for 10 minutes. Triton is quite a strong detergent, so we would not recommend to use it for longer than 10 minutes. You could then continue to use a gentle detergent like Tween in the rest of the buffers, such as the blocking buffer or the antibody dilution buffers, and this will help to keep the cells permeabilized and when you’re washing will help to wash away any excess antibody. So that’s permeabilization.
And then you’re ready for the primary antibody incubation. So, you would dilute your antibody in PBS or TBS buffer containing 0.2% Tween. These are known as PBST or TBST. You can look up recipes for these up on the internet. We would recommend to incubate overnight at 4 degrees. One to two hours at room temperature does very often work but usually the staining can be more specific if you incubate overnight at 4 degrees. The optimal concentration of the antibody will need to be determined. There will be some recommendations on manufacturer datasheets, but you may find you need further optimisation from there. And the aim of that optimisation is really to contain the most contrast between the specific signal and any background staining that you have. And use of a positive and negative control can really help with that. After this step you would wash the cells.
Sometimes primary antibodies are already conjugated. In which case, you won’t need a secondary antibody. But if you do need a conjugated secondary antibody, then this will be your next step. So the secondary antibody would probably be conjugated to a fluorochrome. And we have many of these available, there’s many on the market now, and the most common are for example, FITC, phycoerythrin (PE), Texas red and there are many others as well. As with the primary antibody, we would recommend to dilute these in PBS or TBS containing Tween and perhaps a blocking agent if necessary. This incubation can be for 30 minutes to one hour, possibly longer. Again, this will need to be optimised and it can be done at room temperature. So again, your dilution will also need to be optimised. After this step, again another wash.
So once you’ve finished your staining, you will then want to mount the slides. So covering the cells with the glass on the slide will help preserve the sample, keep the cells safe and also improve the optics when looking you’re looking at it through the microscope. If you are using immunofluorescence, there are some special mountants containing fluorescent anti-fade available and these help protect against photobleaching for when you’re storing the slides for as when you’re viewing them. Many of these special mountants already contain nuclear counterstain such as PI or DAPI. We have many of these at Abcam, for example, we have fluoroshield and brightmount and if you want to try some of them, I can recommend looking these up on the website. So once you’ve mounted your slides, we’d recommend to store them at 4 degrees in the dark to protect some photobleaching. The fluorescence may last up to 2 to 3 weeks or longer, but for good results, we would say to take a look at them as soon as possible under the microscope. And always be aware of any photobleaching while you’re observing and imaging samples.
Before we leave the procedure, I just wanted to mention an unusual tip to you. And that is to use antigen retrieval, which can sometimes be beneficial. Now antigen retrieval is most commonly used of course in immunohistochemistry on paraffin embedded sections and the antigen retrieval is used to break the cross links that are placed there by the paraformaldehyde and this means that the epitope becomes more accessible to the antibody. So, it’s not part of the procedure that’s usually used in immunocytochemistry. However, from some data we have, we know that it can sometimes be necessary to get the optimal results. So, we would say that if our other optimisations been tried, and the results still not satisfactory, particular if you’re still not getting any staining, then you can consider including this step. One we tried in some of our laboratories are to apply heat at 95 degrees in 100 mmol Tris with 5% urea and it had a pH of 9.5 and keep at that temperature for 10 minutes. This means that the proteins are then sufficiently denatured and the antibody binding site is then more accessible. Now you can also check information provided on the datasheets, so very often if we’re aware that antigen retrieval will help then this information will be available on the datasheets. For example, we have an ATP5F1 antibody in the example shown here, so we have staining on HDFn cells. And you can see that without antigen retrieval in the image on the left this is some very different red specific staining. And if you try antigen retrieval, obviously this seems to improve the staining and the antibody has more access to the epitope.
Some what are the advantages of double immunostaining when you have fluorescence. As I said before fluorescent detection gives really good resolution and contrast, so it really lends itself to multiple stains within a single cell line and you could know the localisation and co-localization of two to three or more targets. And really you are only limited by the fluorochromes you use and the fluorescent microscope that you have. So the bottom here there is a couple of images you may have already seen just to show again. So on the left you have oct4 staining in green and actin in red and then we have the cytokeratin in green and actin in red and you really can see the localization of those proteins in this way.
So when you are doing double staining what sort of things do you need to take into consideration? So for each of the target proteins you need to use a fluorochrome that emits at different wavelength so that they are distinguishable from each other. It is good to use conjugated primary antibodies if possible if not you can use conjugated secondary antibodies in which case you will need to select primary antibodies from different host species or they can use different isotypes at times for example IgG1, IgG2a and again this will insure the different types of targeted proteins are distinguishable.
You need to optimize your procedure for each primary antibody individually before you put them together and we really would recommend this. Thus to make sure that reagents are working well and the procedure is working well.