This by not means is the final end-all be-all page for immunfluorescence tips, but it does have answers to Frequently Asked Questions.

Use #1.5 coverslips.

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