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This is the second post in a series on immunohistochemistry (IHC). The first post looked at one of the two main ways of carrying out IHC – paraffin embedded sections – and in this post we’ll take a look at preparing frozen sections.
Frozen tissue section
Depending on what you want to achieve, and what’s available in your lab will affect whether you choose paraffin embedded or frozen tissue sections. For example, paraffin embedded sections are typically better at preserving tissue morphology and allow larger pieces of tissue to be used. However, the use of fixatives like paraformaldehyde can cross link antigens reducing their immune reactivity. Getting them back requires antigen retrieval procedures to ‘unmask’ them, and some antigens can even be irrecoverably destroyed by the preparation of paraffin sections.
The alternative is using frozen sections. Generally, the snap freezing process behind preparing frozen sections is better at preserving antigens, but this may come at the cost of a loss of tissue morphology.
Preparing frozen sections is probably the easier of the two methods.
There are entire textbooks written about IHC, so what follows below is only a guide to help get you started. Fortunately, nearly all antibody manufacturers who have validated their antibodies for use in IHC will have a recommended protocol for you to follow, so whether you are using one from American Research Products or another, please always check the data sheet.
So, without further preamble, let’s get started.
Make sure you tissue is clean. You’ll probably need to change the buffer a couple of times.
Freezing very quickly, rather than slowly letting the sample cool, means only small ice crystals are formed in the sample, as opposed to slowly growing large ice crystals, helping to preserve the tissue and cell morphology.
This will make it easier to handle your tissue when sectioning. You can make a mold out of tinfoil if you don’t have anything specifically designed for it.
Tip: Add a little OCT to your mold first – a few mm worth – then carefully place your tissue section on top, then cover with OCT, making sure the tissue is reasonably central to it.
The OCT should turn white
Again, this step may be slightly different depending on what equipment you have, so not all of this may apply to you – always follow the manufacturer’s instructions where possible.
Tip: Allow the tissue section and mold to equilibrate to -20°C for about 15 minutes before use. This can help prevent the section from fracturing.
Collect the slices on a cover slip
Note: if the specimen is too cool, sections may curl. If too warm, they may stick to the knife.
This helps to fix them to the slides, preventing them falling off during antibody incubations.
You can store slides like this, un-fixed, in a slide box for several months, but if analysis is to be much later, it’s better to store the whole un-sectioned sample until ready.
There are many possible fixing methods, below is an example
This helps block endogenous peroxidase activity
NOTE: it may be necessary to carry out an antigen retrieval step at this point depending on how you fixed your sections.
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, etc.
We hope you’ll have found this protocol a useful start to preparing your frozen IHC sections. As we noted in our previous post on IHC, with so many steps and possible variations in reagents, tissues and antigens of interest, 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