Re: [Histonet] beta-tubulin staining of neurites

From:Kar Ling

I haven't do beta-tubulin staining, but I've done neurofilament staining. I think general procedures apply.
For immunofluorescent staining for neuronal culture, the biggest problem is detachment of neurites.  What I do is to fix them on ice and try not to remove all medium when you change medium.  

Start with neuron culture with culture medium

1. (optional step) withdraw half of the medium and replace with warm phosphate buffer saline pH 7.4 (PBS) . Repeat 2 times. 
2. withdraw half of PBS and add equal volume of ice-cold 8% paraformaldehyde fixation, 10 min, on ice. 
3. Washing with PBS, 5 min, 3 times
4. Blocking with 2% BSA (bovine serum albumin) 0.2% triton-X-100 in PBS, 30 min
5. incubate primary antibody in blocking buffer at 4C, overnite.
6.  Washing with PBS, 5 min, 3 times
7. incubating secondary. (we usually use 1:1000)
8. Washing with PBS, 5 min, 3 times
9. mount and observe.

Karen Ling
University of Southern California
Program in Neuroscience
3641 Watt Way, HNB 209
Los Angeles, CA 90089-2520

----- Original Message -----
Date: Tuesday, August 14, 2007 7:22 am
Subject: [Histonet] beta-tubulin staining of neurites

> 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
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