[Histonet] Help with antigen unmaksing on frozen sections

[Sarah Tarran]

Hi everyone,

I some help with antigen on frozen sections as I am having a lot of
trouble getting antibodies to work. I am using a GFP mouse so can't
formalin fix, parafin embed my samples. I tried frsh freezing but found
that the GFP leaked so I couldn't use that either. So I currently fix my
tissue in 4% paraformaldehyde for 2 hours, then cryopreserve in 18%
sucrose overnight before embedding in OCT the following day. I have tried
non-enzymatic heat retieval (boiling for 15 minutes in Dako's Target
retrieval solution pH 9) but this destroys the GFP, if I use proteinase K
it 'eats' away at the tissue. I have tried diluting it but that hasn't
helped get the antibodies to work. I have also tried overnight at 4 degree
incubations without any luck.

Does anyone have any other ideas that I can try to get the antibodies to
work? The antibodies that I am having problems with are: alpha smooth
muscle actin, LSP-1, procollagen, serum amyloid p and TGF-beta.

Thanks


Sarah Tarran
Postdoctoral Fellow
Vascular Biology Research Centre,
Department of Surgery
Westmead Hospital, Westmead, NSW, 2145