WangLab:Immunostaining General

Protocol for Immunostaining

 * 1) 3X wash of the cells with ice-cold PBS.
 * 2) Fix the cells with 4% paraformaldehyde at RT for 15min.
 * 3) 3x 5min/each wash with PBS in RT.
 * 4) Permeabilize with 0.1% TX-100 in PBS for 15-30 min, at RT
 * 5) Rinse thoroughly with PBS, 3x wash 5min
 * 6) Incubate with 3% goat normal serum or 1% BSA for 20 min, at RT
 * 7) Wash 3x PBS, 5 min/each, at RT
 * 8) Incubate primary antibody (1:100, 10 ug/ml) in PBS at RT for 2 hr or 4oC O/N,.
 * 9) Wash 3X PBS, 5min/each, at RT
 * 10) Incubate secondary antibody conjugated with fluorescence dye (1:200) in PBS, at RT for 1 hr
 * 11) 3x PBS wash, 5 min/each, at RT
 * 12) Mount with anti-photobleaching reagent, seal the slide/dish
 * 13) Imaging