we use the F4/80 antibody from Serotec for both frozen and formalin fixed paraffin sections.
For this antibody the fixation mustn't be longer than 2-3 days in formalin. This is true for an optimal staining for many antibodies, but with the F4/80 it is really obligatory.
I never tried any boosting here because there is always a little bit of background, which we reduced to a minimum by adding mouse serum to the blocking solution.
Pretreatment: 0.2% trypsin for 10 min
We use the primary 1:100 diluted in the blocking solution (10% normal rabbit serum plus 2.5 % mouse serum) , incubation overnight (for frozens 60 min, rt).
As secondary: rabbit anti-rat, mouse adsorbed , Vector
I have to admit that staining of frozen sections is less problematic but we are happy with our staining on FFPE , too.
Good luck !
Antje Marcantonio
Novartis Pharma AG
BU Transplantation Research
Basle/Switzerland
Robert Schoonhoven <rschoon@email.unc.edu>
09.01.2003 13:20
To:
cc: "'histonet@pathology.swmed.edu'" <histonet@pathology.swmed.edu>
Subject: need help F4/80
Mouse IHC folks:
Has anyone had any luck with the rat anti mouse F4/80 (Accurate Scientific)?
This one just doesn't want to work for me on formalin fixed paraffin sections.
In brief I've tried the following:
Pretreatment with trypsin, HEIR citrate, and proteinase
primary dilution's of 1:50, 100 & 500
If anyone has had any good results using this antibody please contact me.
Thanks,
Bob
Robert Schoonhoven
Laboratory of Molecular Carcinogenesis
University of North Carolina