As part of my recent work I had to resolve to PFA fixation followed by sucrose cryoprotection to preserve GFP in spleen tissue sections for IHC. However, i encountered an issue, where tissue sections do not fully transfer onto the glass slide and later appear to crack as the section dries. I was hoping that someone might have encountered the same issue or knows what it may stem from.
The protocol I use is rather straighforward - 4h at 4C in 4% PFA followed by O/N incubation with 30% sucrose also at 4C after which the tissue is embedded in a block of OCT and frozen in the vapour phase of liquid nitrogen.
Can the freezing method be responsible? A lot of PFA/Sucrose protocols use dry ice or methylbutane baths, rather than LN. Alternatively, the 4% PFA was borrowed from a colleague, who apparently makes it by diluting 37% formaldehyde and not by making it up from powder. Is this an acceptable method for making 4% PFA or does methanol present in formaldehyde stock mess with its ability to polymerise?
Any other suggestions are also welcome!
Many thanks,
Vitaly
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