I am doing immunofluorescence of mouse brain cryosections. I collect 25 um sections onto 1% gelatin coated coverslips, let them dry ~30 min, then peel off the OCT, rehdyrate the sections with 0.1 M PB, and proceed with immunostaining. My immunostaining protocol involves permeabilizing and washing with 0.3% Triton X-100 in 0.1 M PB. I do the final washes with just PB to wash the Triton away. After the last wash, I aspirate as much PB as I can and then let the sections dry completely (usually takes ~30 min) before mounting with non-setting glycerol-based media (KPL). My sections always have this cracking pattern. It only occurs when the sections have been permeabilized. I have done tests where I mount sections right off the cryostat or after rehydrating with PB alone and then drying--no cracking occurred. Is the cracking because I let the sections dry completely? That makes sense to me, but why does it only occur after permeabilization? I have tried using charged slides instead of coated coverslips and the cracking still occurs. Other people in my lab don't have this issue and we are following the same protocol that has worked beautifully for the lab for years, including the drying. I also did not used to have this problem, it has only started in the past few months. I have tried several different bottles of Triton and made new PB, which didn't get rid of the cracking. Does anyone know what might be going on?

More details on the tissue handling:

My lab usually drop fixes for 1 hr in 4% PFA and cryprotects in 30% sucrose. That is what used to work for me, but since having this cracking problem and other staining problems (many of my antibodies have stopped working this year), I've tried perfusing with PFA and perfusing/fixing with glyoxal. I've also tried 20% sucrose and 15% or 20% followed by 30%. I've gotten tried premade PFA--did not change anything. After cryoprotection, I freeze the tissue at -80 in OCT or a 2:1 mix of OCT:30% sucrose.