I'm working with 40 micron frozen mouse brain sections. We use a free-floating DAB technique to immunostain for tyrosine hydroxylase, the sections are mounted onto Superfrost plus slides, allowed to air dry overnight and then counterstained using cresyl violet.
The cresyl violet protocol is as follows. Xylenes, 100% ETOH, 95% ETOH, 70% ETOH, Water, Cresyl Violet, Rinse in Water x 2, Differentiation (95% EtOH + glacial acetic acid), 70% EtOH, 95% ETOH, 100% ETOH, Xylenes.
By the time I get to the first water step, the tissue falls off the slides. Others in the lab have used this technique with great success, but I cannot determine why this is happening.
Will pig gelatin coated slides help? Is there something amiss with my cresyl staining procedure? I've considered using cloroform/ethanol in place of the first xylenes to defat the brain tissue, but I was told by a few in the clinical histology lab that my Xyenes, EtOH>water should be sufficient to defat the tissue.
I'd appreciate any suggestions.