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Immunohistochemistry Protocol

Formalin‐Fixed Paraffin Embedded Tissue Sections

IHC

 PDF Version

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

MATERIALS

  • Paraffin‐embedded tissue slides
  • Coverslips
  • Slide racks
  • Staining dishes with lids
  • Plastic slide tray (Baxter Scientific Cat. No. M6304 or similar)
  • Orbital shaker
  • PAP pen
  • Transfer pipettes
  • Deionized water (DI H2O)
  • PBS (Phosphate Buffered Saline)
  • Hydrogen peroxide (H2O2)
  • Primary antibody
  • Biotinylated secondary antibody, HRP conjugated
  • Bovine Serum Albumin (BSA – for blocking)
  • Streptavidin‐HRP
  • DAB
  • Hematoxylin (optional)
  • Acetic Acid (optional)
  • Paramount Coverslip solution

BUFFERS

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 slides in 3% H2O2 for 5 minutes.
  2. Rinse slides twice (2x) in DI H2O for 5 minutes.
  3. Soak the slides in the working citrate buffer and cover with a lid.
  4. Microwave until the liquid boils, about 1‐5 minutes.
  5. Remove from heat and let it stand at room temperature for 20 minutes.
  6. Wash three (3) times for 5 minutes in DI H2O
  7. Remove the liquid (do not touch the tissue!) and use a PAP pen to circle around the tissue.

Blocking

  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 biotinylated secondary antibody to 1:200 in 1% BSA diluent.
  2. Incubate with secondary antibody solution for 30 minutes at room temperature.
  3. Wash slides in PBS three (3) times 5 minutes each on the shaker.
  4. Add enough streptavidin HRP to cover the tissues. Incubate for 30 minutes at room temperature
  5. Wash three (3) times 5 minutes each in PBS on the shaker.
  6. Add enough DAB to cover the tissues. Once the cells start turning brown (inexperienced technicians may wish to observe this under a microscope), wash twice (2x) in PBS for 5 minutes each on the shaker.

Optional Counterstain

  1. Dip the slide rack with the slides into a staining dish of hematoxylin for 30 seconds.
  2. Dip into an acetic bath (200mL DI H2O with one to three drops of acetic acid). Rinse with DI H2O.

Dehydration (Cover staining dishes with a lid in each step)

  1. Dip slides in 70% and 95% alcohol for 3 minutes each.
  2. Dip slides in 2 changes of 100% alcohol for 3 minutes.
  3. Dip slides in 3 changes of xylene or xylene substitute for 3 minutes.

Cover Slips

  1. Drizzle Paramount coverslip solution onto coverslips or slides.
  2. Apply coverslip to slide.
  3. Let the slides dry overnight.