[Histonet] fluorescent labeling of fixed, frozen tissue

[Bower, Jennifer]

Hey everyone!

   I have a big long question, I hope someone out there can help me.
Ultimately, I need to do fluorescent labeling of formalin fixed,
decalcified mouse cochlea. Right now, we are perfusion fixing mice with
10% NBF, dissecting out the bulla with the cochlea still within,
decalcifying with EDTA in the microwave, then freezing the samples and
cutting sections on the cryostat. So far, I have to let my slides air
dry because if I put them straight into PBS, the sections fall off the
slides. Also, everything fluoresces, I suspect due to autofluorescence,
or maybe because of the formaldehyde? Isn't there an easier way? If the
tissue is already fixed, why put the sections into acetone after
cryosectioning? Do I need to do antigen retrieval? Should I try a
pressure cooker method? There is also a lot of superstition surrounding
the use of fluorescents with paraffin embedded tissue, no one seems to
think it will work. Is anyone out there doing any sort of fluorescent
labeling of fixed tissues? Thank you so much for any advice, I feel lost
at this point.

 

Thanks again,

 

Jennifer