Our group is doing OCT frozen section IHC and neurofilament staining for nerve fiber count and density analysis. Unfortunately our rat facial nerve tissue appears to all suffer from severe artifact/fracturing of the specimen. This is making it challenging for us to accurately perform histomorphometric analyses.
Our brief protocol is as follows:
1. Harvest the nerve from the rat and fix in 4% PFA, store at 4 degrees overnight.
2. Nerve transferred to 30% sucrose solution and stored at 4 degrees overnight.
3. Nerves embedded in a cryomold with OCT, frozen with dry ice and stored at -80.
4. Cross-sections at 14 micrometers are obtained with cryotome and placed on slides. We stain the section with toluidine blue to see if that portion of the nerve is suitable; often, at this point, we note some artifact.
5. Application of blocking solution/PBS, primary antibody, incubated at room temp overnight
6. Rinsing with PBS, then secondary antibody, then mounting.
Representative photos are attached. Thanks in advance!
Hello,
Have you considered a FFPE protocol to better preserve your samples? Though it requires more work, generally the samples will be more intact. Cryosectioning is finnicky and a lot can go wrong. Also, you mention leaving antibody on overnight at RT. Is there a reason you don't do this at 4degrees? I'm always very cautious with my BSA solutions as bacteria seems to grow easily in it, especially in non sterile samples such as muscles.
Good luck!
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