We store our brain tissue at -20°C (usually in 50% phosphate buffer, 30% ethylene glycol, and 20% glycerol). Would NaCl in the solution interfere with cryoprotection (i.e., would it allow freezing damage to our tissue)? Would it cause any problems with immunohistochemistry down the line? We want to stain for doublecortin and Ki67.
Thank you!
Hi Angela!
We use 30% sucrose diluted in PBS in order to cryoprotect the whole brain tissue. Then we freeze brains with isopentane and store them at -80 ºC until the day of processing. Then we do cryostat section and preserve tissue sections at -20 ºC in cryoprotectant solution (a little different than the used before, sodium phosphate 25 mM (pH: 7.3), 30% ethylene Glycol and 20 % Glycerol).
Hope this work for you.
PS: Were these brains obtained by perfusion with PFA or freshly obtained? If were obtained by perfusion, you have to take into account the time of post-fixation. Usually, if you plan to do IHC you have to let a post-fixation of around 4h. If you let an overnight post-fixation you could lose some specific signal.
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