[Histonet] Frozen sections and loss of nuclei?

From:"Tan, MinHan"

Message
Hi there Histonetters,
 
I would=20like to seek your advice about a problem I am having with frozen sections.
 
I am working on some frozen tissue dating back to about 1998, and performing IHC on parathyroid tissue with p27 antibody. I use the DAB-ABC method.
 
I have noted that certain samples (and particular samples only) exhibit loss of nuclei in the central region (an amorphous blob of tissue extending to near the margins, about 70-80% of the specimen) (no staining with hematoxylin either). Any nuclei=20that stain with my IHC in that area are very faint, and there is no hematoxylin counterstained nuclei in the entire region. Nuclear staining seems to be fine near the margins - of course it can be argued that these=20are artifacts. Where there are folds of tissue, both nuclear and background staining is prominent. Other samples of the same type of tissue seem to stain OK throughout the tissue. No problems with paraffin-embedded tissue at all, the nucleus stains fine in every sample I have tried.=20
 
It is unlikely to be due to underfixation, since I immerse my 5 micron unfixed slide in neutral buffered formalin for 30 min prior to IHC. I see it regardless of=20 whether I perform HIER on my frozen sections or not. (yes, I do perform HIER on my frozen sections)
 
I wonder if the following explanations are plausible:
(a) tissue has degraded - but it=20would not make sense for degradation to take place from inside out.
(b) sections may be too thin for some reason? (mine are 5 micron) - but this might explain why only the borders and folds stain.
 
Thank you!

Min-Han Tan
Van Andel Research Institute

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