[Histonet] beta-tubulin staining of neurites

[cbass <@t> bidmc.harvard.edu]

Hello Everyone,
 
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:
 
SHS-Y5Y cells
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?
 
Thanks everyone!
 
Caroline