I have a bunch of precious samples that appear underfixed for immunofluorescence with a particular antibody. They are all in frozen OCT blocks now. Should I melt them down and re-fix them or can I just post-fix the tissue sections on slides for 1 hour or longer?
Cheng-Ying Ho
Immerse the tissue in 70% ethanol three times for 30 minutes each at room temperature. Immerse the tissue in 90% ethanol two times for 30 minutes each at room temperature. Immerse the tissue in 100% ethanol three times for 30 minutes each at room temperature.
Complete protocol is given here. Please go thru
https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-ihc-staining-paraffin-embedded-tissue#:~:text=Immerse%20the%20tissue%20in%2070,minutes%20each%20at%20room%20temperature.
Kashif Ameer Isn't this the rehydration step for deparaffinization of FFPE tissue? My tissue is not paraffin embedded.
Cheng-Ying Ho
The deparaffinization is needed to remove the layer of paraffin but in your case you do not need the re-hydration step as tissue is not paraffin embedded in your case.
Thawing-refreezing cycles usually contribute to protein degradation and increased unspecific staining and artifacts. At this point your best option may be to cut the tissue using a cryostat, mount the tissue, post fix it and stain on slides. For unfixed frozen tissue, post-fixation might need some troubleshooting, since different epitopes are more easily fixed. A mixture of 50% methanol/acetone at -20 C for 20 min works for some samples ( https://www.cellsignal.com/learn-and-support/protocols/protocol-3589-if) or for 15 min RT with 4% formaldehyde (https://www.cellsignal.com/learn-and-support/protocols/protocol-if) If your protocol requires the proteins to be crosslinked. Remember that prolonged fixation might mask the epitopes and thus you might need to perform an antigen retrieval.
Luis Leon We routinely do post-fix with 4% paraformaldehyde for 15 min RT but it does not help much. I wonder if we can do up to an hour or even longer.
Cheng-Ying Ho depending your immuno, you can push the post-fixation for a longer time. I have already gone up to 45 minutes post-fixation in cold PFA 4% or 2% without any problem (keep them in ice for protein preservation). But each antibody is different. These parameters (time and concentration) should be tested on fresh-frozen random samples if you are not sure. In this case you can use the proper protocol for your precious sample. Good luck !
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