What is IHC?What IHC controls should I set up before I plan the experiment?

IHC stands for Immunohistochemistry. Immunohistochemistry is a technique for localizing and visualizing an antigen in a tissue section by using an antibody specific for the target antigen.
The immunohistochemistry procedure consists of tissue preparation, antibody incubation, and a series of detection reactions. Technically, when the procedure is performed on cells (cell smears, cytocentrifuge preps, etc.) rather than tissue sections, it is termed immunocytochemistry, but in practice the two terms are often used interchangeably.

What IHC controls should I set up before I plan the experiment?

A set of controls should be run with the antibody and tissue to be tested. These controls are for initial testing only. More controls may be needed for troubleshooting purposes.

Negative controls: without primary antibody, without secondary antibody, and without streptavidin-enyme conjugates.

Positive controls: a positive antibody with the test tissue, or the test antibody with a positive tissue.

Why does my negative control show strong signal?

The signal is due to non-specific cross-reactivity of detection reagents.

Negative Control	Possible Cause of Signal	Solution
Primary Ab only is omitted.	The secondary Ab is binding non-specifically to the tissue.	Add 0.1% tissue-specific serum to the secondary Ab. sDilute the secondary Ab. Change species of secondary Ab.
The secondary antibody only is omitted.	The streptavidin-enzyme conjugate is binding non-specifically to the tissue.	Block tissue with the Avidin/Biotin Blocking Kit (Cat. No. BK-1311-06).
The streptavidin-enzyme conjugate only is omitted.	Intrinsic tissue enzyme activity is interfering with the reaction.	Treat tissue with hydrogen peroxide solution (for HRP) or add levamisole to substrate (for Alk Phos).

Why does my positive control have no signal?

In most cases, this is because the antibody is not optimized or the tissue is not adequately treated.

Positive Controls	Cause for No signal	Solution
Using an antibody known to react with the test tissue, or using the test antibody with cells known to contain the antigen.	Fixatives may have reduced access of antibody to antigen.	Perform microwave target retrieval procedure or protease digestion.
	Ab may be too dilute.	Titrate Ab to determine theoptimum dilution that gives the best signal-to-noise ratio.
	Secondary Ab does not recognize the primary Ab.	Titrate Ab to determine theoptimum dilution that gives the best signal-to-noise ratio.
	The enzyme/substrate system is defective or incompatible.	This can be confirmed by performing the Dot Blot Test (see instuction manual).

What references could you provide if I want to learn more about IHC?

Carson, F.L. (1990) Histotechnology: A Self-Instructional Text. ASCP Press, Chicago.

Elias, J.M. (1990) Immunohistopathology: A Practical Approach to Diagnosis. ASCP Press, Chicago.

Taylor, C.R. and Cote, R.J. (1994) Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist.

W.B. Saunders Co., Philadelphia.

Shi, S.R., Gu, J., Kalra, K.L., Chen, T., Cote, R.J., and Taylor, C.R. (1995) Antigen retrieval technique: a

novel approach to immunohistochemistry on routinely processed tissue sections. Cell Vision 2: 6-22.