Four-color IHC staining protocol

Date Published: July 18, 2023

The protocol described below is a sequential staining process and may be adapted to any desired number of colors by adding or removing the antibody/detection/chromogen step. Use care with selection of antigens and incubation times when multiple colors are applied to one slide. Addition of extra blocking step may be performed between antibody applications, but is not necessary. This protocol is for 4-color staining of formalin-fixed paraffin-embedded human tonsil with mouse monoclonal anti-human macrophage (CD68), rabbit polyclonal anti-human lambda right chain, rabbit polyclonal anti-human kappa light chain, and mouse monoclonal anti-human cytokeratin (high MW).

 

Reagents required:

IHC Tissue Primer (ADI-950-234), Antibody Blocker/Diluent (ENZ-ACC108), IHC Wash Buffer (ADI-950-235), IHC Antigen Retrieval Reagent pH 9 (10X)(ENZ-ACC113), POLYVIEW® IHC Reagent (mouse-HRP)(ADI-950-112), POLYVIEW® PLUS AP (anti-rabbit) reagent (ENZ-ACC110), HIGHDEF® DAB Chromogen/Substrate SetENZ-ACC105), HIGHDEF® Blue IHC Chromogen (AP)(ADI-950-150), HIGHDEF® Red IHC Chromogen (AP)(ADI-950-140), HIGHDEF® Yellow IHC Chromogen (HRP)(ADI-950-170).

 

Reagents preparation:

Some reagents are stable for up to one week.  For best results, prepare all reagents fresh.

  1. 1X HIGHDEF® DAB Chromogen working solution: Add 40 µL of HIGHDEF® DAB to 1 mL of HIGHDEF® DAB Substrate buffer in a mixing bottle. Mix thoroughly. Working solution is stable for at least two weeks if stored at 4°C.
  2. 1X HIGHDEF® Blue IHC Chromogen (AP) working solution: Add 1 drop (approximately 20 µL) of HIGHDEF® Blue chromogen to 1 mL of of HIGHDEF® Blue buffer in a mixing bottle. Mix thoroughly. Working solution is stable for 6 hours if stored in the dark.
  3. 1X HIGHDEF® Red IHC Chromogen (AP) working solution: Mix equal volumes of HIGHDEF® Red chromogen and HIGHDEF® Red buffer in a mixing bottle. Mix thoroughly. MAKE FRESH. WORKING SOLUTION IS STABLE FOR ONLY 20-30 MINUTES.
  4. 1X HIGHDEF® Yellow IHC Chromogen (HRP) working solution: Add 1 drop (approximately 20 µL) of HIGHDEF® yellow chromogen to 1 mL of of HIGHDEF® Yellow buffer in a mixing bottle. Mix thoroughly. Working solution is stable for one day if stored in the dark.

 

Staining procedure:

NOTE:  Wash slides with 1X IHC Wash Buffer after each step except after background blocking step.

1.        Antigen Retrieval

a.        Perform antigen retrieval (if necessary). Recommended solutions: IHC enzyme antigen retrieval reagent (Prod. No. ADI-950-280), Antigen Retrieval Reagent, pH 6 (10X) (Prod. No. ENZ-ACC112), or IHC antigen retrieval reagent (EDTA, pH 8.0) (Prod. No. ADI-950-273).

2.        Tissue Preparation

a.        Incubate slides for 5 minutes with IHC Tissue Primer. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

b.        Block for 5 minutes with Antibody Blocker/Diluent.

3.     Staining Part 1

a.     Apply mouse monoclonal anti-human macrophage (CD68). Incubate for 30 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

b.     Apply mouse-HRP PolyView™ IHC Reagent solution and incubate for 20 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

c.     Develop the HRP-mouse antibody with 1X HIGHDEF® DAB Chromogen working solution for 5 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

4.     Staining Part 2

a.     Apply rabbit polyclonal anti-human lambda light chain. Incubate for 30 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

b.     Apply POLYVIEW® PLUS AP anti-rabbit IHC Reagent solution and incubate for 20 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

c.     Develop the rabbit-AP antibody with 1X HIGHDEF® Blue IHC Chromogen (AP) working solution for 5 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

5.     Staining Part 3

a.     Apply rabbit polyclonal anti-human kappa light chain. Incubate for 30 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

b.     Apply POLYVIEW® PLUS AP anti-rabbit IHC Reagent solution and incubate for 20 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

c.     Develop the rabbit-AP antibody with 1X HIGHDEF® Red IHC Chromogen (AP) working solution for 5 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

6.     Staining Part 4

a.     Apply mouse monoclonal anti-human cytokeratin (high MW). Incubate for 30 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

b.     Apply POLYVIEW® HRP anti-mouse IHC Reagent solution and incubate for 20 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

c.     Develop the HRP-mouse antibody with 1X HIGHDEF® Yellow IHC Chromogen (HRP) working solution for 5 minutes. Wash slides with three changes of 1X IHC Wash Buffer for 1 minute each wash.

7.     Counterstain

a.     Apply either Hematoxylin (e.g. HIGHDEF® hematoxylin, Prod. No. ENZ-ACC106) or Methyl Green (less time and dilution needed for Methyl Green). Incubate for 2 minutes.

8.     Post Processing

a.     Rinse the slides with distilled or deionized water.

b.     Blot excess water from the slide without letting tissue specimen dry. Make sure tissue is wet prior to mounting.  DO NOT dehydrate the slides in alcohol and xylenes.  

c.     Coverslip slides with 2-3 drops of permanent mounting medium, HIGHDEF® IHC Mount (Prod. No. ADI-950-261).

 

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