Dr. Giorgio Cattoretti
Associate Professor of Clinical Pathology
Institute for Cancer Genetics, Columbia University
New York, USA

Alkaline Phospatase (AP) or Peroxidase (HRP)?

Answer:

Routine IHC is done with peroxidase (HRP) and DAB development, an insoluble, light insensitive, brown pigment which well contrasts with the blue nuclear counterstain of Hematoxylin. Can be mounted permanently with xylene-based mounting media, with excellent optical charateristics.

Red development of AP has even greater color contrast with Hematoxylin counterstain, however with most development, you should use an acqueous mounting.

Consider your tissue first:
Hematopathology specimen are traditionally done with AP, because of high endogenous peroxidase in BM and blood. Beware of cells with high endogenous Acid Phosphatase (AcPh; e.g. macrophages): if the pH of your development is off, you may stain endogenous AcPh along your exogenous AP.

• If your tissue has levamisole-resistant endogenous AP (e.g. gut, bone), use HRP, unless you boil it (see below).
• If your tissue has been exposed to high heath (MWO), your endogenous enzymes except HRP are gone.
• If your tissue has high endogenous peroxidase (e.g. BM), use AP.
• If your tissue has endogenous biotin, barely quenched by blocking systems (e.g. kidney, liver), use a non-biotin-based system with AP.

You can use sodium azide (NaN₃)-containing solutions with AP-conjugates. You should not, whenever you have HRP.