Immunocytochemistry (ICC)

•Transfection of cells seeded in 6 well plates with coverslips

•Media was decanted

•Washed thrice with PBS (5 min each)

•Fixed using 3%PFA (para formaldehyde) for 10 min

•Washed thrice with PBS (5 min each)

•Permeabilized using 0.01% Triton X 100 for 15 min

•Washed thrice with PBST (5 min each)

•Blocking in 5% NGS overnight at 4ºC or at RT 1-2hrs.

•Incubated in 1º Ab for 2hrs

•Washed thrice with PBS (5 min each)

•Incubated in 2º Ab (in 0.2% NGS) conjugated to Fluorochrome for  1hr.

•Washed thrice with PBS (5 min each).

•5µl of DAPI was added on corning slideand coverslip was placed on it upside down such that the cells are towards the slide

•Extra DAPI was removed and sealed using transparent nail enamel.

•Visualized under Fluorescence or Confocal Microscope