IHC Protocol

IHC identifies the expression pattern of a protein in tissue through specific antibody binding. So the tissue used for IHC is often preserved and fixed to retain localization of the protein. The protocols included here are mainly for these formalin-fixed, paraffin-embedded tissues (IHC-P), which offer superior tissue/cell morphology, but require antigen retrieval before staining. In addition to the general protocol, we also include here the step-by-step mouse-on-mouse and human-on-human IHC protocols.

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IHC for paraffin-embedded tissue

Solutions and reagents

Protocol

Solutions and reagents

1. Xylene

2. Ethanol, anhydrous denatured, histological grade (100%, 95%, 85%, 75%)

3.Washing buffer:

1XPBST	1 L
10X PBS	100 mL
dH2O	900 mL
Tween-20	1 mL

10X PBS	1 L
NaH2PO4•2H2O	2.84 g
Na2HPO4•12H2O	27.2 g
NaCl	90 g
dH2O	1000 mL

The pH should be about 7.2. Adjust if necessary with 1 M NaOH or 1 M HCl

4. Antigen Retrieval Solution:

A. 10mM Sodium Citrate Buffer, pH 6.0

C6H8O7•H2O	0.21g
C6H8Na3O7•2H2O	0.21g
dH2O	1000 mL

Adjust pH to 6.0

B. 10mM Tris Buffer with 1mM EDTA,pH 8.0 or 8.5 or 9.0

Tris	1.21 g
EDTA	0.37 g
dH2O	1000 mL

Adjust pH* to 8.0 or 8.5 or 9.0

*Note: Please refer to the antibody for individual antigen retrieval buffer and working conditions.

5. 3% Hydrogen Peroxide

6. Hematoxylin QS

7. Permanent Mounting Medium

Protocol

A. Deparaffinization and Rehydration

Heat slides in an oven at 60 °C for 5 min.
Wash slides 3 times for 10 min each in xylene.
Wash slides in 95% ethanol, 1 min.
Wash slides in 85% ethanol, 1 min.
Wash slides in 75% ethanol, 1 min.

Rinse slides for 5 min in distilled water.

B. Antigen Retrieval*

Put the slides in Antigen Retrieval Solution and keep 120℃ for 2.5 min in pressure cooker.
Then cool down at room temperature.

*Note: Please refer to the antibody for individual antigen retrieval buffer and working conditions.

Wash slides three times with distilled water (2 min each).

C. Staining

Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 15 min.

Wash slides twice with 1X PBST (2 min each).

Dilute primary antibody in the IHC Antibody Diluent per recommendation on the data sheet.
Apply primary antibody to each section and incubate 90 min at room temperature*. Make sure the primary antibody solution covers the tissue evenly.

*Note: Please refer to the antibody for individual buffer and working conditions.

Wash slides three times with 1X PBST (2 min each).

Apply to each section secondary antibody and incubate for 15 min at room temperature*.

*Note: Please refer to the antibody for individual buffer and working conditions.

Wash slides three times with 1X PBST (2 min each).

Add freshly prepared DAB substrate to the sections.
Incubate tissue sections with the substrate at room temperature until suitable staining develops (generally about 5 min).

Rinse sections with water.