Need troubleshooting in immuostaining - (Jun/27/2011 )
Hi, all, I am new in immunostaining. Right now I am doing double-immunostaining of fresh adult rat cardiomyocytes. The problem is that I get no fluorescence except outside of cells. Proteins of interest are either in the inner cell membrane or cytosol. I use 1/1000 Laminin to stick to cells and 4% formaldehyde to fix cells in 30 mins. The concentration of primary antibody and secondary antibody (Cy3 and Cy2) are 1/100 and 1/500 respectively. I need your suggestions! Thanks!
Did you permeabilise (incubate in 0.1-0.05% tween 20 in TBS or PBS) the cells before adding antibody?
bob1 on Tue Jun 28 01:20:36 2011 said:
Did you permeabilise (incubate in 0.1-0.05% tween 20 in TBS or PBS) the cells before adding antibody?
I incubated cells with PBS with 0.1% Triton to permeablized them
That should work fine. It could be autofluorescence of the cells you are seeing, which is supposed to be reduced by using Bouin's fixative (20% DMSO, 10% H2O2) after fixing with formaldehyde. How much did you wash the cells after fixing?