[Histonet] Help with antigen unmaksing on frozen sections

Joanne Mauger MAUGER <@t> email.chop.edu
Wed Feb 11 07:17:24 CST 2009


Try fixing your cut sections in 10%NBF for 20-30 minutes after
deparaffinizing, then do the retrievals. SMA doesn't usually require any
retrieval. TCR beta works well with pepsin for 20 mins. - this is less
harsh than proK. I'm not familiar with the other abs you mention.

Jo Mauger

>>> "Sarah Tarran" <sarah_tarran <@t> wmi.usyd.edu.au> 02/10/09 10:08 PM
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
that the GFP leaked so I couldn't use that either. So I currently fix
tissue in 4% paraformaldehyde for 2 hours, then cryopreserve in 18%
sucrose overnight before embedding in OCT the following day. I have
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
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
incubations without any luck.

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


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

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