Re: Dilution buffers?

From:Kathleen Spencer

Whatever our blocking solution is, that is usually what we make our 
primary in. Our secondary is usually made in PBS. But, some protocols 
call for different dilution buffers. Depends on the the antibody.

On Wednesday, July 30, 2003, at 07:16  AM, Anna-Karin Robertson wrote:

> Hi!
> I have a very general question about dilution buffers for primary and
> secondary antibodies. What is normally used? I have looked at several
> protocols and see no clear or common principle.
> I am staining, and double staining, on mouse tissues with primary
> antibodies, usually rat or hamster, and sometimes these antibodies are
> biotinylated. I use normal immunhistochemistry or immunofluorescence. 
> If I
> have a goat-anti-rat as a secondary antibody I block with goat serum 
> first,
> before the primary antibody iss applied. If I have a avidin-labelled
> secondary step I block with BSA before applying the primary antibody.
> I know that a lot of people use BSA or dried milk in their dilution 
> buffers,
> but if I am using a secondary step being a streptavidin- or
> avaidin-conjugated fluorochrome, I realize I must avoid anything 
> containing
> biotin, i.e. serum (?), milk, BSA (?) etc in order not to lower or 
> block the
> binding. Should avoid this in the dilution of the primary antibody as 
> well?
> But if I use only PBS, won't I risk getting I high background? The 
> antibody
> diluent from immunohistochemistry from Pharmingen seem to contain only 
> and sodium azide, though.
> Is there a difference between what is applied in immunofluorescence 
> compared
> to normal immunohistochemistry?
> I realize each specific case must be tested out, but I would be very
> grateful for some general advice.
> Anna-Karin Robertson
> Karolinska institute
> Stockholm, Sweden

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