IF Two Antigen Retrival Steps - (Jan/31/2017 )

I have an interesting scenario. I'm focused on the colocalization of two proteins via IF. Protein #1 performs much better with the 20 minute pH 6.0 citrate buffer antigen retrival method, protein #2 performs much better with the 20 minute Tris-EDTA pH 9.0 method. Is it possible to do two antigen retrival methods, one after the other? Has anyone done this?

I haven't done it, but I would be concerned that you could damage the first antigen by performing the second recovery. However, both methods you are using there are relatively mild, so I would guess it will work. I guess your only option is to try it and see.

I gave it a shot, I did the pH 9.0 20 min boiling first followed by the 20 min boiling in pH 6.0.

5% of the sections look amazing, the other 95% have no signal. Very inconsistent staining. Do you think it has to do with uneven heating in a domestic microwave or was too much protein degraded?

can you try a more uniform heating method (eg hot plate)?