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Immunohistochemical staining is used to identify specific constituents in tissue sections or immobilized cells.
To detect the reaction site, the antibody/antigen complex is labeled with an enzyme that can be reacted with a suitable substrate to give a colored product. Proper fixation is crucial to successful staining. Formaldehyde fixation is often used as the routine initial method of choice for tissue and cell fixation. The antigen (usually immobilized cells or tissue) is fixed and adhered to a glass microscope slide. A primary antibody reacts with the immobilized antigen to form an antigen-antibody complex. A second, biotinylated antibody specific for primary antibody reacts with the complex. Streptavidin conjugated to Peroxidase reacts with the ab-ab-ag complex immobilizing the peroxidase at the site of the antigen. Finally, substrate is added causing a colored precipitate to form on the slide at the location of the antigen. This slide is analyzed using a light microscope or embedded and prepared for electron microscopy.

Reagents Required

PBS Wash Buffer. Phosphate Buffered Saline (PBS). Use 10X PBS, pH 7.2 (0.2 M Potassium Phosphate, 1.5 M NaCl) Code # MB-008. Dilute appropriate volume to 1X with deionized water.

Formaldehyde Fixative. Dilute to 4% in PBS buffer.

Antibody Dilution Buffer. Prepare 100 ml of PBS Wash Buffer supplemented with 1 ml of normal serum of same species as the host used for the secondary antibody. See page 74 for a listing of control serums.

Biotinylated Secondary Antibody. Prepare dilution of biotinylated secondary antibody in Antibody Dilution Buffer. Use biotinylated secondary antibody conjugate against the same species as the primary antibody. See page 34.

Streptavidin Peroxidase. Prepare dilution of Streptavidin Peroxidase (code S000-03) in PBS buffer. See page 63.

DAB Substrate. Use DAB (code DAB-10). See page 58 for a complete listing of substrates.

Hematoxylin Counterstain and Mounting Media.


1. Grow cells on glass microscope slides, glass coverslips or slide culture chambers. Remove culture medium and gently wash cells 3 times with ice cold PBS. Fix cells by adding a volume of 4% formaldehyde in PBS equal to the original volume of culture medium for 30 minutes on ice. Remove the fixative and wash 3 times for 5 minutes each with PBS. If desired, incubate 5 minutes in 1% H2O2 in PBS to remove endogenous peroxidase activity. Wash the fixed cells 3 times for 5 minutes each with PBS.

2. Prepare appropriate dilution of primary antibody by diluting in Antibody Dilution Buffer. Remove the buffer from the cells. Add a sufficient volume of diluted primary antibody to cover the cells. Incubate with primary antibody for 60 minutes at room temperature. If the primary antibody has a low-affinity for the antigen, incubate at 4° C overnight. Remove primary antibody solution. Wash 3 times for 5 minutes each with PBS.

3. Remove the buffer from the cells. Add Diluted Biotinylated Secondary Antibody and incubate for 30 minutes at room temperature. The optimum dilution may be lot specific. Refer to back cover inset for recommended dilution ranges. Remove solution. Wash 3 times for 5 minutes each with PBS.

4. Remove the buffer from the cells. Add Diluted Streptavidin Peroxidase and incubate for 30 minutes at room temperature. Remove solution. Wash 3 times for 5 minutes each with PBS.

5. Remove buffer. Add DAB substrate and incubate approximately 10 minutes or until sufficient color develops.

6. Remove solution. Wash 3 times for 2 minutes each with distilled H2O. Counterstain with hematoxylin for 1 to 5 minutes depending on the concentration and color intensity desired. Wash 3 times for 2 minutes each with distilled H2O. Dehydrate the cells with 100% ethanol 4 times for 2 minutes each. Clear the cells with xylene 4 times for 2 minutes each. Add 2 -3 drops of Mounting Media, add coverslip and allow to air dry.

7. Observe cells under the microscope. A positive reaction should be visible as a brown precipitate. The nuclei should appear light blue.


1. Immunocytochemical Methods and Protocols. L.C. Javois, ed. Methods in Molecular Biology series Volume 34. Humana Press, 1994.

2. Antibody Techniques. V.S. Malik and E.P. Lillehoj, eds. Academic Press, 1994.

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