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Sequntial double staining with same species primaries

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rajkumar:
Hi,

I am planning to do sequential double staining with two rabbit primary antibodies. I was told that after adding the first primary and secondary I could block any free binding sites with rabbit IgG. But wouldn't this Rabbit IgG then bind the second anti-rtabbit secondary?

Any help would be appreciated

Vin

immunoem:
Hi Vin,
check out the following web link from Jackson labs. Should help calrify the procedure:
http://www.jacksonimmuno.com/technical/fab-blok.asp
Good luck.
Ian

rajkumar:
Hi Ian,

It did! Thanks.

Jossy:
Hi!     I did sequential double IF with two anti-rabbit primary Ab, with donkey secondary Ab.  I used donkey serum to block in both IF (i finished "first" IF, then block 30 min with 5% DS and incubate with the "second" anti-rabbit primary Ab)
With this protocol, I don't see any background or cross reaction between both primary Ab. (one was a nuclear marker and second was a citoplasmic marker).
I was listened about the Fab blocking step, but i don't have money to buy it, and decided try this way.
I hope this help.
Good luck  :D

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