Unusual Non-specific Binding in Immunofluorescence - (May/30/2012 )

Hi everyone.

I've been having some issues with unusual background in my Immunofluorescence experiments.

I am currently targeting a viral protein with a very good mouse monoclonal antibody, and I am hybridizing it with Alexa594 goat anti-mouse. I initially block my fixed, permeabilized cells with 1% donkey serum in PBS for 30 minutes, and I dilute my antibodies in 1% BSA in PBS. I wash 3x in between staining. Unfortunately, I am getting bright red dots all over the cell that looks like this in my mock infected cells (file attached below).

My infected cells look good (viral protein localizes in the nucleus but there is some background in the cytoplasm)

Is anyone familiar with this unusual background?

I tried antibody dilutions, increased number of washes/incubations, and different washes (PBS-T, PBS, NBCS/PBS), but to no avail...i still get this unusual problem. I even tried secondary staining by itself...and i did not get the background, which suggests that something is wrong with the primary step....

HELP!!!

Have you tried titrating the primary antibody for this method?

yes i have tried titrating both the primary and secondary antibodies. i begin to lose my target protein signal. i even tried secondary conjugate by itself and I got no fluorescence. this suggests that something is wrong with the primary step. i even tried blocking with 10% Normal donkey serum in PBS-T today instead of 1%, and I still got background in my mock infected cells.

i also found that there were very small white/peachy mush in my stock primary antibody...is this a problem?

bkbk on Thu May 31 00:40:03 2012 said:

I decided to compare the particular antibody with another antibody that targets another viral protein, and I found that the latter gave me clean results in mock infected cells. So in conclusion, something is wrong with the former antibody.

hopefully the new antibody i ordered last night will work