Re: rat on mouse immunos
|From:||Cathy Gorrie <C.Gorrie@unsw.edu.au>|
I have always used a 5% solution of the serum from the host animal of
the secondary antibody. This is applied to the tissue for 30 mins
before incubation in the primary. You don't need to rinse it off.
Just shake or wipe off the excess.(See list archives for discussion
on the merits of these different techniques)
The idea behind this is that this serum will contain any number of
antibodies that will bind to other antigen in your tissue, thus
preventing cross reactivity later down the track.
Eg rat anti-mouse mac-3 is your primary, swine anti-rat might be your
secondary. therefore I would use 5% normal swine serum (made up in
your usual diluent) as the blocking agent.
The only problem would be if there was a stray antibody in the serum
that unfortunately binds to your target antigen ie mac-3 but this is
unlikely as primary antibodies now are highly specific and serum
antibodies are generally not.
As well, if you are using DAB/HRP systems, you will need to block for
endogenous HRP in the tissue, which is mainly found in blood vessels,
liver to name a few. This is accomplished by incubating in a 3% H2O2
in PBS solution prior to other staining. The H2O2 reacts with any
endogenous HRP in the tissue so it won't react when you come to do
the DAB reaction at the end.,
Hope this helps, Cheers Cathy
At 3:49 PM -0500 28/3/01, Rita Angel wrote:
>I have an immuno question for the animal research people out there. I have
>a mac-3 antibody which is rat anti-mouse. I will be staining mouse tissue.
> What kind of blocks do you use to block endogenous staining? Our normal
>block to use is 0.5ml of 2% mouse serum in 25mls of secondary. I rotate
>this for 1 hr. to allow binding and then use at the normal time (10 min.)
>Does anyone else have any other procedure that they use? This did not work
>the last time I used it.
>Thank you for your input,
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