1 reply to this topic

[rugermatt]

Hi running the risk of a dumb question here.  I am doing Immunocytochemistry on mouse cells grown on chamber slides.  The question is, When is it ok to use blocking serum, primary antibodies, and secondary antibodies of the same species?
For instance I am using mouse cells probing with ProteinX anti-mouse and a secondary that is Goat anti-mouse conjugated to 488 (alexafluor probe).   It is my understanding it is ok to use Goat serum to block for this protocol.

However in a similar experiment probing with ProteinX anti-Goat (with documented mouse cross reactivity) and a secondary that is Rabbit anti-Goat conjugated to 488.  If I use Goat serum to block I get a disturbing amount of background.

So my question is the Goat serum used to block tagging tons of stuff with Goat Igg and then Goat 488 recognizing all of that.  And if so should I try to block with serum from the species that my primary was raised in to reduce non-specific signal resulting from the species Igg?  I’m very confused and I appreciate any help with this issue. Thanks

[mdfenko]

you should use normal serum from the species in which the secondary antibody is prepared (or you could use bsa or milk to block).

you should use rabbit serum to block when probing with proteinx anti-goat.

Edited by mdfenko, 28 September 2010 - 11:49 AM.