Basic Protocol for antibody labeling of cultured cells:
1) 1.) Plate cells on number 1.5 coverslips
2) 2.) Rinse culture media off with PBS or media without serum to remove serum proteins. Rinse means replace liquid with the same amount of wash solution. No incubation necessary. You might want to move the coverslips to a 6 or 12 well dish for labeling for ease of solution changes.
3) 3.) Add 4% paraformaldehyde in PBS, incubate at room temp. for 3-5 minutes
4) 4.) Rinse 3x with PBS or PBST (PBS + 0.1% triton-x100, with 0.5% BSA)
5) 5.) Incubate in PBST for 30 minutes prior to adding primary antibody diluted in PBST
6) 6.) Incubate with primary antibody either 1 hour at room temp, or overnight at 4 degrees C. You can add multiple primary antibodies together if they are from different species.
7) 7.) Rinse 3x with PBST
8) 8.) Add diluted secondary antibody (e.g. molecular probes goat anti-mouse Alexa 488 at 1:500) to the coverslips and incubate at room temp for 1 hour. You can add multiple antibodies together as long as they are directed against different species.
9) 9.) Rinse 3X with PBST
10 10.) If you want DAPI labeling and your mounting media does not have it already, add DAPI at 90mg/ml and wait 15 minutes prior to mounting
11 11.) Pick up coverslips with a forceps and touch them to a kimwipe to wick off excess PBT, put them face down on about 20-50 ml of Prolong gold, Fluoromount G or slowfade mounting medium.