Immunofluorescence Protocol
4%-PFA Fixed Cell Line Slides


Do not let the tissues dry out once they are re‐hydrated.
Use separate tubs for antibodies and negative control slides to avoid contamination.


  • Coverslips
  • Slide racks & tray
  • Staining dishes with lids
  • Orbital shaker & Transfer pipettes
  • Deionized water (DI H2O)
  • PBS (Phosphate Buffered Saline)
  • Triton X‐100
  • Primary antibody
  • Fluorescent secondary antibody
  • Bovine Serum Albumin (BSA – for blocking)
  • Glycerol

Permeabilize Membrane (Optional)

  1. Add one drop of PBS/0.1% Triton X‐100 to each well to permeabilize the cells. Incubate slides for one (1) minute at room temperature.
  2. Remove the liquid and wash the slides twice (2x) in PBS, 5 minutes each on the shaker.


  1. Remove the liquid and add 5% BSA into each well. Incubate overnight at 4°C in a humid chamber.

Primary Antibody

  1. Dilute the primary antibody to the recommended concentration in 1% BSA diluent.
  2. Remove BSA and incubate with primary antibody for one (1) hour at room temperature.
  3. Remove primary antibody solution and wash slides three (3) times in PBS, 5 minutes each on the shaker.

Secondary Antibody and Detection

  1. Dilute the biotinylated secondary antibody to 1:200 in a solution of 1% BSA diluent.
  2. Remove fluid and incubate with secondary antibody for one (1) hour at room temperature in dark place.
  3. Wash three (3) times 5 minutes in PBS on an orbital shaker. Remove excess fluid.

Cover Slips

  1. Add several drops of coverslip solution (50% glycerol / DI H2O) to the slide.
  2. Place coverslip on top of the slide and store slides at 4°C until ready to view.