I would like to know if there is a difference between the time and temperature of the antibodies incubation during IF protocol, e.g.:
Primary Antibody: 1 hour in room temperature or 4*C overnight?
Secondary Antibody: 2 hours in room temp. or at 37*C?
This time, I incubated the first for 1 hour in RT and the second for 2 hours in RT too. When I saw the result, the second antibody fluorescence was too low and I could only see the bright cells using a 40x magnification.
My goal is to see if 2 transfected proteins are co localized, but the problem is that the "protein A" has a GFP tag (pEGFP-Protein-C3) and the protein B, an HA tag, forcing me to use a primary and secondary Ac.
The GFP tag work beautifully but fight with glow of the secondary Ac. Even if I want tho see a co localization, in this way, I will only see The Green!
What do you guys think?
I will appreciate any help!