[Histonet] Using antiserum for IHC

[Martin S.]

Hi All,
 
Has anyone got some tips for using antiserum for immunolabelling frozen
tumour sections? I have some anti asialo GM1 (rabbit polyclonal, reacts
with moues NK cells). There are a couple of reports of it being used to
label frozen sections but they dont give any details. I have tried it at
1/10, 1/50, 1/100, 1/500, 1/1000 and 1/5000 dilution but for all
dilutions there just seems to be background staining of the whole tissue
(it does get progressively better with increasing dilution but even at
1/5000 it wouldnt be good enough to sepatae positive staining from the
background.
I used a biotinylated secondary (goat anti-rabbit from Vector, which
works well with another rabbit primary antibody and is clean when used
without primary) and blocked with 5% normal goat serum before the
primary incubation and then just with PBS in between antibodies. I used
Vectors ABC kit with DAB for detection.
 
I havent used antiserum before and was wondering if there are any
special considerations or blocking steps that I should try. The
secondary is only anti-rabbit IgG wheras the antiserum is polyclonal so
maybe thats the reason - however in one of the previous reports they
used an anti-rabbit IgG as secondary...........
 
Any suggestions welcomed!
 
Sonya