Immunofluorescence Protocol
for Tissue Sections


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


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


Working Citrate Buffer

  • 9mL of 0.1M Citric Acid (10.5g citric acid monohydrate to 500mL DI H2O)
  • 41mL of 0.1M Sodium Citrate (14.7g sodium citrate dehydrate to 500mL DI H2O)
  • 450mL of DI H2O

Deparaffinization and Rehydration (Cover staining dishes with a lid in each step)

  1. Dip slides in three (3) changes of xylene or a xylene substitute for 3 minutes each.
  2. Dip slides in two (2) change of 100% alcohol for 3 minutes each.
  3. Dip slides in one (1) change of 95% alcohol for 3 minutes.
  4. Dip slides in one (1) change of 70% alcohol for 3 minutes.
  5. Rinse slides twice (2x) in DI H2O for 5 minutes.

Antigen Retrieval (Microwave Method)

  1. Soak the slides in the working citrate buffer and cover with a lid.
  2. Microwave until the liquid boils, about 1‐5 minutes.
  3. Remove from heat and let it stand at room temperature for 20 minutes.
  4. Wash three (3) times for 5 minutes in DI H2O
  5. Remove the liquid (do not touch the tissue!) and use a PAP pen to circle around the tissue.


  1. Apply enough 5% BSA with a transfer pipette to cover the tissues.
  2. Incubate the slides overnight at 4°C in a humid chamber.

Primary Antibody

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

Secondary Antibody and Detection

  1. Dilute the fluorescent secondary antibody to 1:200 in 1% BSA diluent.
  2. Incubate with secondary antibody solution for 1 hour at room temperature in dark place.
  3. Wash slides in PBS three (3) times 5 minutes each on the shaker.

Cover Slips

  1. Apply 50% glycerol/PBS to the middle of the slide.
  2. Apply coverslip to slide and store slides at 4°C until ready to view.