[Histonet] immunocytochemistry for Systems biology

[vinay sj]

Dear Histonetters,

I am working on Systems biology project for the quantification of some phospoproteins using a phospho-specific antibody. As is the case with life in general, there are problems. So, I need your expert advice.

My experimental protocol: fix with 4 % Paraformaldehyde, permeabilize with 0.2 %Triton, Block with goat serum, stain with primary antibody and secondary antibody( raised in goat).

>From flow cytometry experiments, I see that there is a background staining when i do the immunocytochemistry without applying my stimulus(i.e pfa+ triton+antibodies). From western blots, I know that without stimulus, there is no phosphoproteins and hence the phospho signal ought to be zero. The background staining is 5 fold higher than plain autoflourescence(pfa+triton) and 3 fold higher than autoflourescence due to secondary antibody(pfa+triton+secondary antibody). My phospho signal is around 4 fold higher than my background signal.


 I do not understand how to eliminate the robust primary antibody nonspecific staining. I do not want to use methanol, since I would like to do the experiments in 96 well format as well. 

I have read that aldehyde fixation could give rise to non specific signals. But I want to stick to Paraformaldehyde, since I cannot cool my 96 well plates, to use methanol.

Any help will be appreciated.

Regards,
Vinay.