Secondary goat antirabbit and antimouse conjuagated to alexa showing fluorescenc - (Jul/29/2015 )

hi,

Iam working on a cervical cell line and want to do immunofluorescence for certain endogenous proteins. When I did the procedure, I found that the negative control was showing fluorescence. So I repeated the experiment again with IgG only. The groups were

1. cells without IgG

2.cells containg ig G only

3. cells with secondary conjugated to alexa 488

4. cells with IgG and secondary.

In this I found it was the secondary that bound to cells to give a background fluorescence. How can I reduce this.

The expt conditions were. fixing in 4% formaldehyde 10 min,

permeablization with triton X100- 0.1% 5 min

blocking in 5% goat serum 1 hr

primary in goat serum overnight at 4 or 2hr at RT 1:200 dilution

secondary goat serum 1:1000

I dont know where the mistake in the procedure is.The cells do not show autofluorescence. Suggestions are welcome

jaya

How much washing do you do and what conditions (concentrations of salt, detergent etc.)?

I wash it 3-4 times with PBS and I dont use any detergents to wash it on cells

Try adding somewhere in the range of 0.01 - 0.5% Tween-20. The detergent makes the wash more stringent. You can also try using a higher salt concentration (e.g. 200 mM rather than the current 150 mM).