I am staining mouse skin cryosections (10um) with PanH3, an antibody that binds to histone 3. When harvesting this tissue I embed in OCT and freeze using dry ice and liquid nitrogen. When viewing under a a fluorescent microscope, these sections appear to be significantly distorted and damaged even when no AGR (0.5% SDS) or fixation (0.5% PFA) is applied. The epidermis looks completely garbled and the dermis contains webby, hazy areas. I have applied DAPI to a few slides w/o going through my staining protocol and they seem fine so I am sure my sectioning technique is ok. Can anyone explain what I'm seeing (image attached)?
I do not fix my tissue before OCT embedding or use sucrose. I post-fix in 0.5% PFA at the beginning of the staining procedure.
Hi Ben, I don't think your freezing method is responsible for the appearance of your sections. I think maybe something is going wrong when you cut them....maybe the cryostat anti-roll plate or the blade angle are not optimally adjusted.
Best wishes,
Martin
Hi Martin thanks for your response. I think that may be possible, however my other slides that haven't underwent my staining procedure look very good when stained just with DAPI (with respect to section integrity and cellular morphology). I now suspect that my issues lie with my post-fixation I.e 0.5% PFA is too low. Typically 5% is used. I will soon be runnig this experiment again using 5% PFA and fresh sections.
Increasing the concentration of post-fix from 0.5% to 4% PFA seemed to do the trick. I suspect that when I used 0.5% PFA the sections were unable to withstand the harsh SDS AGR I subsequently applied.
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