Protocols for Immunohistochemistry

Invitrogen staining kits are provided with complete protocols for use. This page lists the steps involved in using selected Histostain™ kits and the time required for each step. It also contains protocols for removing endogenous peroxidast, digesting tissue and antigen recovery.

Removal of Endogenous Peroxidase Activity

  • Procedure 1: (Use for paraffin-embedded sections. Not for frozen sections.) Prepare peroxidase blocking solution by adding one part 30% hydrogen peroxide to nine parts methanol. Submerge slides in this solution for 10 minutes. Wash three times with PBS.
  • Procedure 2: (May be used with frozen or paraffin-embedded sections.)


Treat slides with Peroxo-block™ for 45 seconds. Wash immediately.

Removal of residual biotin, avidin or streptavidin activity

  • Residual Biotin: Incubate section with the avidin solution from Invitrogen's blocking kit. Wash. Incubate with the d-biotin solution from the kit. Wash thoroughly.
  • Residual Avidin or Streptavidin: Incubate section with the d-Biotin solution from Invitrogen's blocking kit. Wash thoroughly.


Heat Induced Epitope Retreival
The epitope retrieval procedure is used to reverse the loss of antigenicity that occurs with some epitopes in formalin-fixed, paraffin-embedded tissues. We have found that some antibodies require Antigen Recovery (e.g., estrogen receptor, and Ki-67 antibodies), while the staining of many other antibodies will be enhanced by Antigen Recovery (e.g., S-100, cytokeratin, and synaptophysin antibodies). No licensing fee is required for using Antigen Recovery. Please call (1-800-955-6288) or e-mail our Tech Service department if you have any questions.

A. Materials
  • Hot plate
  • 1 liter glass beaker
  • 0.01 M citrate buffer, pH 6.0 (No. 00-5000)
  • EDTA Solution (Cat. No. 00-5500 for antibodies that require EDTA instead of citrate buffer)
  • PBS

B. Protocol
  1. Mount tissues should on silane or poly-L-Lysine coated slides, or slides coated with HistoGrip™
  2. Deparaffize slides and, if desired, perform avidin/biotin blocking and endogenous enzyme quenching (see above). 
  3. Wash slides with distilled water 3 times for 2 minutes each. 
  4. Put the slides in a slide rack and place it in a 1 liter glass beaker (Pyrex) containing 500 ml of 0.01 M citrate buffer. 
  5. Place beaker on hot plate. Heat the solution until it boils and keep it boiling for 10 minutes. 
  6. After heating, remove beaker from the hot plate and allow it to cool down for at least 10-20 minutes at room temperature. 
  7. Rinse slides with PBS and start the immunostaining protocol.

Tissue Pre-digestion

Tissue digestion recommendations for all predilute and 50x concentrated IHC antibodies are given in our Anatomical Pathology Catalog. Tissues should be mounted on slides using HistoGrip™, Para-Pen , Fro-Pen, silane, or poly-l-lysine.

  1. Deparaffinize, rehydrate, and quench endogenous enzyme activity (if necessary).
  2. Wash slides with distilled water 3 times for 2 minutes each.
  3. Incubate sections with proteolytic enzyme at 37oC. Incubation time should be determined for each application (for digestion reagents standardized for 10 min incubations, inquire about Digest-All™
  4. Rinse slides with PBS 3 times for 2 minutes each and continue the immunostaining procedure.

Procedure for selected Histostain™ kits

Histostain-SP and Histostain-SAP Kits

  1. Prepare slides and controls, and perform peroxidase blocking step if necessary. 
  2. Incubate section for 10 min. with Serum Blocking Solution. Drain and blot away excess. 
  3. Incubate with user-supplied Primary Antibody (30-60 min. at room temperature is usually sufficient). Wash. 
  4. Incubate for 10 min. with Biotinylated Second Antibody. Wash. 
  5. Incubate for 10 min. with Streptavidin-Enzyme Conjugate. Wash. 
  6. Incubate for 5-10 min. with Substrate-Chromogen mixture. Wash. 
  7. Counterstain with Hematoxylin and mount with Aqueous Mounting Solution.