Immunohistochemistry and Immunocytochemistry

Immunohistochemistry and immunocytochemistry are powerful techniques for localizing the molecular expression of proteins in tissues and cells, especially when combined with the in situ hybridization technique. Immunohistochemistry and immunocytochemistry techniques are primarily considered to be qualitative measurements, but when used together with a computerized imaging program, staining distribution and intensity can be semiquantified. This chapter describes immunochemical techniques routinely used in our laboratories for protein localization in tissue sections and cultured cells with the representative staining shown in Figs. 1 and 2 . The protocols are based on an indirect immunoperoxidase detection via the avidin:biotinylated-peroxidase complex method with 3,3′-diaminobenzidine as the chromogen, a popular method suitable for bright-field microscopy. The first immunohistochemical protocol described is intended for paraffin-embedded tissue sections and the second immunocytochemical protocol is intended for cultured cells. It is highly recommended that, before beginning the protocol, the operator read Subheading 4 .   Fig. 1.  Immunohistochemical localization of endothelial cell nitric oxide synthase (ecNOS) and angiotensin II type 1 receptors (AT 1 -R) in the ovine placental artery from a representative ewe at 142 days’ gestation. Dark cytoplasmic color indicates the positive staining. Dark nuclear color is hematoxylin counterstaining. Tissue sections were incubated with mouse antibody against ecNOS (2.5 μg/mL; upper panel) or rabbit antibody against AT 1 -R (2 μg/mL; bottom panel). An adjacent section was used for controls, replacing the primary antibody with an equivalent amount of mouse (for ecNOS) or rabbit (for AT 1 -R) IgG protein ( see figure inserts ).   Fig. 2.  Immunocytochemical localization of ecNOS and AT 1 -R in ovine feto-placental artery endothelial cells. Dark cytoplasmic color indicates the positive staining. Dark nuclear color is hematoxylin counterstaining. Endothelial cells were incubated with the primary ecNOS (2.5 μg/mL; upper panel) or AT 1 -R (2 μg/mL; bottom panel) antibody. The endothelial cells in adjacent chambers were used for controls, replacing the primary antibody with an equivalent amount of mouse (for ecNOS) or rabbit (for AT 1 -R) IgG protein ( see figure inserts ).