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Put simply, immunohistochemistry (IHC) is a way of staining tissue samples to reveal if and where a specific protein is located in that sample.
That one sentence makes it sounds somewhat easier than the reality. The reality is there are so many possible combinations of tissues types, ways to prepare tissues, species, antibodies and detection systems that many consider IHC as much art as science.
But that’s OK, but because we’re here to help you, and once you’ve got a protocol optimizing for your application, they can be rock solid – working time after time.
IHC-P refers to the staining of tissues that have been fixed (usually in neutral buffered formalin) and then
embedded in paraffin before being sectioned.
Image 1: A paraffin embedded tissue block
IHC-Fr is where the tissues are frozen and then sectioned – a subject we’ll tackle in a post in the near future.
What follows below is a simple protocol for paraffin embedded section for you to build on, with some hints for things to look out for. You’ll likely need to optimize the protocol to fit with the reagents and tissue types you’re using, but this should help get you on the right track.
This is one of the most important steps, as you want to ensure that you preserve the tissue correctly keeping the tissue structure, localisation of antigens and the physical nature of the antigens themselves so that your antibody can detect them – paraformaldehyde is used to help achieve this, fixing the tissue.
Swish it around to clean the sample. Change the buffer a couple of times if need be.
You’ll need an excess of paraformaldehyde in this step, as a rule of thumb go for a volume about twenty times greater than the tissue. Excess will be fine.
We’ll do this step-wise, by immersing in increasing concentrations of ethanol, all for an hour at 4°C, and then dimethylbenzene.
Allow to cool at room temp until the paraffin sets, then store at +4°C.
Tip: Pour a small layer of paraffin into the mold, about a couple of mm, then add your tissue sample, then cover. This allows the tissue to be surrounded by a few mm of paraffin on all sides.
Now, when you’re ready, prepare your tissue sections:
Image 2: A microtome
This step is important, as without it, it’s likely that your sections won’t stain properly or only very poorly.
Place your slides in a rack and then wash as follows:
The tissue fixing step forms methylene bridges in the sample, cross linking proteins which can mask antigen binding sites. As such, antigen retrieval is needed to render antigens ‘visible’ to detection and allow immunohistochemical staining.
There are two main methods for doing this, Heat induced Epitope Retrieval (HIER) and enzymatic, and multiple version of them. Use only one
Some antibodies will specify the antigen retrieval method. If so follow such guides, otherwise, below are two example protocols:
Add enough to cover the slides by a few cm, in a non-sealed vessel (boiling will cause evaporation and pressure to build). Don’t let the slides dry out, so watch for evaporation or boiling over.
If using a domestic microwave, set to full power and wait until the solution comes to the boil, if using a scientific microwave, follows the manufacturer’s instructions.
It’ll be hot be careful not to spill or burn yourself!
This lets the slide cool enough to be handled and the antigenic epitopes to re-form after the high temperature treatment
The exact process may depend on the enzyme you are using (e.g. pepsin, proteinase K, trypsin), therefore, follow the manufacturer’s instructions for preparation (including buffers) and use.
Ensure you use enough water to cover the slides.
Longer incubation with the enzyme may be needed, depending on enzyme, but too long may ‘over-retrieve’, damaging tissue morphology or increasing background later on
As always, if your antibody has an included protocol for IHC staining, follow that first. If not, the below is a guide to get you started, but you will likely have to optimise it for your antigen, species or tissue type.
Image 3: Different magnifications of paraffinized intestinal tissue
We hope that this will give you a good place to start. As we said above, with so many steps and possible variation in reagents, IHC can be as much art as science.
In the future we’ll be offering up more posts on trouble shooting IHC, to help you get the best result possible.
Filed Under : IHC