This by not means is the final end-all be-all page for immunfluorescence tips, but it does have answers to Frequently Asked Questions.
Permeabilization necessary for molecules to have access to cell interior.
Do secondary antibody controls.
Two standard protocols from lab I was in 2010 to 2013. I'm surprised they only use 2% PFA because back in the day I learned 3.7 to 4%.
Fix with 2% PFA in bilayer buffer 10 minutes RT Permeablization with 0.01% Triton 1 min (10% stock diluted 1:100) in bilayer buffer blocking 1/2 hour casein in bilayer buffer staining as needed
Fix 2% PFA in PBS for 10 to 20 minutes RT Permeabilize 0.1% saponin in PBS for 5 to 10 min Wash PBS 3X optional to quench autofluorescence: Glycine 50mM in PBS for 10 to 30 min Wash PBS 3X block 1/2 to 1 hr 10% FBS Wash staining as needed- primary antibody in 2% FBS in PBS Wash PBS 3X secondary antibody