I'm looking for a decent protocol for staining beta-tubulin in tissue culture. Basically, we are doing a neurite extension assay in chamber wells, where the neurites extend onto the filter in culture and then we stain the neurites for beta-tubulin. We use the following reagents:
beta-tubulin antibody, rabbit polyclonal, Cell Signaling, Cat #2146
Alexafluor 488, goat anti-rabbit, invitrogen Cat # A11008
We are new to immunofluorescence so any help would be appreciated. We are trying this out initially in cells plated in regular 24-well plates. The best we've seen is a light diffuse staining with 1:500 of the primary and 1:2000 of the secondary. My guess is that both dilutions have to be cut down. Any suggestions? Also, should there be a permeabilization step? I think we tried tween-20 in one step. Do we need triton-x?
Histonet mailing list