some IHC staining problems - (Aug/15/2006 )
I cut 10um thin frozen tissue section for IHC. I have a number of problems during IHC stain. They happen randomly and I don't know what's the reason and how to prevent them:
1. After cutting, the biopsies did not happily stick onto glass slides. One end of the biopsies sticked to the blade and the other end flied up towards my glass slide. So when I force my glass slides close, the biopsies fold and piled together rather than forming a nice thin layer on the slides.
2. During IHC, I found the thin sections kept losting during ethanol fixing and antibody washing steps. So there was no intact biopsies left after the whole IHC processes.
3. After either HE or IHC stain, I found epithelial cells are there but lumen cells are gone.
And, one day I didn't well look after my slides. When I'd incubated primary antibody for 1hr and about to wash out the primary, I found the slides are dried. I continued with the rest of the IHC procedures anyway, but I'm not sure whether I can trust the staining results or not?
Any suggestion is welcomed.
If the cut sections dont stick to ur glass slides, check if its right to use them as some slides cannot hold slices and u will lose pieces of the tissue as u proceed with the IHC. U need special slides.
If the slices fold or pile, it happens for no obvious reasons, u could try adjusting the temp. setting in the cryostat. make it more colder, might help.
if ur slides have dried up, I would repeat the whole thing on a new slide.
In addition to the advice from scolix, you may need to pre-coated the slides with poly-L-lysine, so that the tissue section can stick to the slide.
Hope this help.
Hope this help.
i agree with that but DONT FORGET TO WASH 5 times or so with pbs as free poly amino acid is toxic to cells.