Hello Everyone! I have searched the archives because I am sure this is a problem that has been encountered before, but I have been unsuccessful in finding the right thread. I ran an IHC series dilution on a new primary antibody for NeuN. I am using 35 um rat brain sections that were cut on a cryostat and stored in Cryoprotectant until ICC began. After finishing the ICC protocol, I mounted the slides that were floating in phosphate buffered saline onto slides that I subbed two weeks ago (.5% gelatin subbing). I allowed the slides to dry from Friday afternoon until Tuesday afternoon. I tried to dehydrate them for coverslipping and when they went into the dH20 bath, some of the sections began to fall off of the slides. This continued in the 50% Alcohol bath. All tissue that made it through those first two baths in the series stayed on throughout, but I did lose quite a few sections, especially those that had the lowest concentration of primary antibody. My experience has been that highly concentrated primaries make the tissue a bit sticky, but the best staining generally comes from the least concentrated. Does anyone know what the problem might be? I think it might be a subbing problem but I'm not sure. Thanks in advance.
Amanda Madden Research Assistant P.S. The subbing protocol that I used only called for one "dip" into the gelatin solution, and didn't call for any type of acid wash. Also, our cryostat is a Leica CM3050S and I was wondering if any of your labs have established a working range in terms of humidity. Our lab has been experiencing very high humidity levels (up to almost 80%), so we were wondering if anyone has established that cutting cannot be done unless the humidity is under x%. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet