I am undertaking immunohistochemistry on sections from a knockout mouse line we have. The issue I am having is that in the final steps of the protocol, a number of ethanol baths (50%, 70%, 95%, 100% and 100% for 3 minutes each) are undertaken, followed by placing the sections on the slides and a xylene bath for 15-20 minutes. I complete the first of the 100% ehtanol baths and the sections are coming out very dry, I would describe them as crispy even. This causes problems as they are not then sticking to the slides when placing them in the xylene. When trying to bypass the xylene step and just mounting the coverslip, the sections end up getting crushed and crumble making them unusable. Has anyone else come across this problem and if so what were their solutions to it?
Yes the sections are not fixed to the slide and the solutions are getting behind and lifting them off.
Go find a very old histology book for precise instructions but you can dip the slides in a solution of nitrocellulose dissolved in Ether this coats the slides so the sections cannot move then move through the alcohols and the xylene will remove it at the end. This was often used with silver methods as they were notorious for lifting sections from slides.
For the future you need to use slides with some kind of coating....Mary
Thanks Mary! The slides I am using are charged though and thus should this not assist in the binding and effectively serve the same function as the nitrocellulose? It is my mistake for not mentioning it but in the protocol our lab employs, we wash the sections while they still sit in the wells and it is not until after the final ethanol bath and prior to the xylene bath that we try to mount them on the slides. If I understand correctly, you are suggesting mounting the sections prior to the alcohol washes?
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