I have a HA-Glut4-GFP tagged construct that I have transfected into my cells. I intend to visualize after insulin stimulation without permeabilisation so that I can just detect the exofacial HA-tag on the membrane. I'd like to compare 2 protocols - (1) with fixing first, then detection with anti-HA primary antibody and flourescent-conjugated secondary and the (2) to label first with anti-HA primary antibody and then fix etc. My question is how long do I do the labeling for on the 2nd protocol before fixing? I was thinking for 1h on ice - is this long enough?

Thanks

Suman