[Histonet] any suggestions?

[Till, Renee]

Another tech who does not have much experience with histology came to me
with questions about his immunos. They are doing IHC with various cd
markers on frozen sections of mouse aorta. He has encountered
particularly strong background(or so he's been told, he thinks it is
actual staining) with one of the antibodies that was made in rat. He is
using a goat anti-rat F(ab')2 from Jackson as the secondary. It is not
absorbed against mouse. I have asked all about his dilutions and
incubations times, but he doesn't seem to think that is the problem. I
gave him an avidin/biotin block to try and see if that helps. Any other
ideas? I am not familiar with cd markers myself. The only problem I
could find just in talking to him was that he was blocking with rabbit
serum? I told him you normally match your block with the host of the
secondary, but would that make that big a difference as far as
background is concerned?  Would the fact he is using frozen sections
have anything to do with it? Or could it just be the stain? I know they
are doing cd54, but I'm not sure if this is the one he is having a
problem with. 

I know this is not much informations, but I would still appreciate any
input

Thanks,

Renee'


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