Our cryoprotectant recipe calls for 50% phosphate buffer, 30% ethylene glycol, and 20% glycerol. We accidentally used phosphate buffer saline (with the ethylene glycol and glycerol) instead. I have three questions:
1) Will the NaCl interfere with cryoprotection (i.e., will it allow freezing damage to the tissue)?
2) What would freezing damage look like?
and 3) What problems could this mixup cause later with IHC?
Further details: we store our tissue at -20°C and want to stain for Ki67 and doublecortin.