I got the following from a grad student here at Penn State. I am not sure how
to solve his problem if possible. Does anyone have any suggestions I can
forward?
Roberta Horner
Animal Diagnostic Lab
Penn State University
"I am having some difficulties sectioning mouse tumor samples for
immunofluorescent analysis. We originally went the OCT route because we are
staining for T cell markers and were worried that the heating that occurs
during paraffin embedding would compromise the T cell receptor. The samples
are a little old, but we are hoping to section and stain for immune cell
infiltrates. When sectioning with the cryostat, the tissue and OCT is quite
brittle and the sample is not intact enough to transfer to a slide. Two
colleagues have given the following suggestions:
1. Thaw the OCT blocks, remove the tissue, and place in a new block with fresh
OCT media. I've tried this technique on a few practice samples and the new OCT
media seems to be less brittle and I'm able to get tissue on a slide, however
the tissue itself still seems to be poor with either freeze or sectioning
artifacts.
2. Thaw the OCT blocks in water, remove the tissue and place in formalin
overnight, place in 70% EtOH, then paraffin embed. Section on a microtome.
Check the fluorescently labelled antibody data sheet to see if paraffin
embedding interferes with binding. Try to stain and see what happens.
I was hesitant to try the second suggestion because I have found no protocol
that takes tissue originally stored in OCT blocks and subsequently redirects
them to formalin and paraffin for microtome sectioning. If you have any
recommendations on how to move forward and section these difficult samples, or
know anyone at the diagnostics lab or at Penn State that could help, that would
be much appreciated!"
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