IHC Related Problem
Sudheendra Rao N R
(by sudhee26 from gmail.com)
Thu Dec 20 23:47:37 EST 2007
Hi StewJW, Nick
thanks for the suggestions.
Actually as we told, we have also tried other company anti-GFAP, anti-S100b
(we used anti-s100b because it can selectively stain glial cells..not like
GFAP..immature neurons might express GFAP..so with the former we expect only
glial cells..but in negative control we are seeing neurons!!!--pyramidal in
cortex and hippocampus)
when we use primary we dont get this pattern..its only when we use secondary
As Nick was pointing out it cant be said that our secondary is free from
interacting with rat antibodies.
We use vector biotinylated secondaries and we have tried a different lot to
end up with same results. We can however try with vector elite ABC kit..
PS: we have use Fluorescein Conjugated( Rat Adsorbed )antiMouse..but same
results in Negative Control!! that rules out antirat mouse antibodies in
we would say..its not the rat antibodies the secondary is interacting
with.it some rat protein which has homology to mouse protein..hence
antimouse is binding to it..not quite sure..
We tried 10% blocking horse serum, Primary 1:1000 and secondary 1:2000
dilution..but same results in the negative control.
Can any one tell me how does a secondary antibody interact..as in through Fc
How do antiMouse, antiRabbit, antiGoat secondaries differ??
Sudheendra and Deepti.
On 12/20/07, StewJW <stewjw from gmail.com> wrote:
> Could it just be your primary antibody causing the problem? Zymed
> (Invitrogen) sells a primary mouse anti-GFAP antibody, catalogue
> 08-0021, which comes in a range of concentrations to work with your
> own development system or you can use one of Zymeds kits, which have a
> low background and very high sensitivity, The Histostain Plus Kit is a
> peroxidase DAB kit for mouse primary antibodies, catalogue 85-9143.
> These kits use a biotin labeled secondary antibody followed by a
> peroxidase labelled strepatavidin. The reagents come in dropper
> bottles and no reconstitution or pipetting is required, and they are
> designed specificially for IHC.
> Link for primary antibody product note:
> Methods mailing list
> Methods from net.bio.net
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