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

Negative Controls:
	Labeled antibody/MouseBlock mixture is omitted. Labeled antibody/MouseBlock mixture & avidin-enzyme conjugate are omitted. Primary antibody is replaced with an isotype matched non-reactive antibody.
Positive Controls:
	Primary antibody is replaced by an antibody known to react with the test tissue.

Why does the negative control have staining?

Insufficient blocking of endogenous biotin.
Bacterial contamination of water or reagents.
Excessive adhesive on slide.
Over-development of substrate.
Tissue is necrotic.

Why do the slides have no staining?

Reagents applied in wrong order or step omitted.
Tissue may be overfixed or improperly fixed.
Incorrect mounting medium.
Tissue dried out during staining procedure.
Incorrect primary antibody dilution.
Incorrect target retrieval procedure.

Why do the slides have weak staining?

Wash buffer not adequately drained after every wash step.
Inadequate incubation times.
Substrate/chromogen defective or contaminated.
Primary antibody overdiluted or may need longer incubation times.
Improper fixation or deparaffinization of tissue.
Low concentration of antigen.

Why do the slides have excessive background?

Inadequate rinsing of slides.
Reagents not properly diluted.
Over-development of substrate.
Incomplete deparaffinization.
Tissue dried out during staining procedure.
Inadequate blocking of endogenous biotin.
Inadequate blocking of endogenous peroxidase.
Diffusion of soluble antigen.
Primary antibody concentration too high.

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