Direct Immunofluorescence Protocol - (Jul/06/2006 )

In the next couple weeks I will hopefully begin immunofluorescence to confirm functional expression of various receptors of interest which will be transfected into adherent HEK293 cells. I've been searching for various pointers on direct immunofluorescence but only found generalized tips and skeletonized protocols for indirect immunofluorescence. Does anyone out there know of a protocol outlining direct immunofluorescence to study transfected receptor expression? The Sigma-Aldrich protocol that I have doesn't entail a blocking step, prompting me to ask if this step is necessary, especially for direct immunofluorescence. If blocking is vital, what solution should I block with? I've read that the best method is to use serum from the host of the antibody. Thanks so much for reviewing my questions. I look forward to receiving some responses...

I think blocking is vital and reduces background considerably. We used to block with goat or calf serum. Some time even horse serum.

I am not familiar with direct immnunofluorescence. U could contact companies which provide antibodies to give u a protocol.

direct IF is basically the same as an indirect with the difference that your primary antibody is attached to a flouorphore, so you don't have to add a secondary antibody.
For direct IF I use the same protocol as in an indirect immuno but after washing the primary you are done!

As for the need of the blocking step, it varies for different samples and antibodies. I generally include it when I'm working on tissue and omit it when the IF is on cultured cells, because I've very low background.

I make the blocking solution with 10% goat serum, 2% bovin serum albumin (BSA) and 0,4% Triton X-100 and incubate with it for 60 minutes. I really don't know anything about the species thing you mention, I always use this same recipe and work with mouse and rabbit antibodies. It makes more sense though to use different serum and antibody species, so they won't react between them.


Here's a link for you: http://www.emsdiasum.com/microscopy/techni...letterBSA-C.pdf
(it's commercial, but useful)

hope I've helped

i learnt IF in a cheap lab - we used fish skin gelatin to block on cultured cells (from sigma) - I would always block JIC