Superfrost Plus Slides

[joshuaallen07]

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. 

[excalibur]

Several things may be going on.

You may have a a bad batch of slides or they have been open too long.

Thick sections do not adhere as well as thin sections.

Do not touch the slide where you will put the tissue. Oils from your hand or hand lotion will coat the slide so tissue will not adhere. Always pick up and handle the slide by the label end.

[joshuaallen07]

Several things may be going on.

You may have a a bad batch of slides or they have been open too long.

Thick sections do not adhere as well as thin sections.

Do not touch the slide where you will put the tissue. Oils from your hand or hand lotion will coat the slide so tissue will not adhere. Always pick up and handle the slide by the label end.

Thanks for your reply.  I guessing it was a bad batch of slides/slides that have been open too long as I never touch the surface of the slides.  I spend a week troubleshooting the issue and using pig subbed slides and replacing the first xylenes step with 100% chloroform helped with my problem.  Who knew something that should have been so simple would take a week to troubleshoot and fix? 

Thanks again!

[MDeng]

simply rinsing the slide in PBS instead of water can help keeping the tissue on the slide