I am looking at staining mouse brain tissue for three different proteins (TPH-1, FFAR 2, and IL-6). The tissue is fixed (24 hours in paraformaldehyde twice, and then 2 rounds of 24 hours in sucrose with sodium azide after that) and mounted onto slides after being cut to 20 micrometers on a cryostat. I’ve run into a wall when it comes to seeing if I would be able to do a non-fluorescent IHC stain for all of these proteins given that they are pre-mounted. Most articles I have read through involve free-floating tissue samples at various other thickness levels, which doesn't help me and the post-doc in my lab doesn't know if she can give me any for sure ideas on if it is okay to do the IHC. We haven't started slicing yet, as I want to confirm that my process would be able to continue with an IHC stain. I am also planning on working with RNAscope, which is why I would either need to slice at 20 or 40 micrometers as there are procedures for those two thickness levels.
If I am able to use different stains on the same slides that would be great, since I only have so many brains to work with.
Hopefully this all make sense, but I am free to answer any follow up questions if anything isn't certain.
It sounds like you can do counterstaining on your pre-sliced IHC tissues. You just need to see if your antibodies are IHC quality, and host aminals of these antibodies are all different: rabbit, mouse, & goat etc. You also need to have secondary antibodies which are also different fluorophores conjugated for these different hosts.
Please check the attached article. Hope this helps.
If you want to perform chromogenic IHC on your slides, please check the following link:
https://sysy-histosure.com/ihc-p-guidelines/multiplex-ihc-p
However, to me, your frozen section might fit for fluorescent multiplex IHC rather than chromogenic IHC.
20 microns is a little thick for complete antibody penetration of a mounted CNS tissue section. (The floating section method would allow penetration from both sides.) You might also find that all of the steps and rinsing for a 3 color chromogenic IHC protocol will detach a large percentage of the sections. Permeabilizing the section (detergent) would also increase section loss. It’s frustrating, but I suggest a test run with one antibody. I’ve watched one of my students lose 80% of her frozen spinal cord sections with a 2 antibody localization.
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