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....
Edited by bkbk, 29 May 2012 - 10:37 PM.