Immunohistochemistry Protocol

Immunohistochemistry protocol for anti-5-methly Cytosine (5-mC) and  Bromodeoxyuridine (BrdU) Polyclonal Antibodies

Product numbers: P00704, P00013

Please see reference citations for individual products for more recent product application data.

Original antigenic studies performed by Dr David Stollar, Tufts University, Boston, MA

ABSTRACT

New methods for double and triple colour labeling using monoclonal antibodies to the proliferation-associated markers 5-methly-cytosine, BrdU and Ki67 are described. In order to make incorporated 5-methyl-cytosine or BrdU accessible to most antibodies, mild denaturation of the DNA is needed, and this is usually obtained by exposing the cells to acid or base.  This procedure destroys most cellular antigen, including nuclear TdT and Ki67.  In this study, we show that fixation in cold methanol instead of 70% ethanol for 30 minutes followed by immersion in 7 x 10-3 N NaOH for 10-15 seconds allows BrdU staining with the simultaneous detection of nuclear cytoplasmic and membrane assigns as well as preservation of morphological detail.  This method is optimal for detection of nuclear Ki67 and TdT.  These reagents, together with antibodies to membrane assigns can be included in triple colour labeling using second layers conjugated to FITC, TRITC and colloidal gold.  With these methods it is now possible to characterize the phenotype of dividing cell populations such as precursors in central lymphoid tissues and germinal centre blasts in peripheral lymphoid organs.

Reference for tissue staining with anti-BrdU

• Campana D, Coustan-Smith E., Janossy G., Department of Immunology, Royal Free Hospital School of Medicine, London, UK. J Immunol Methods 107(1):79-88 1988 Feb 24  Double and triple staining methods for studying the proliferative activity of human B and T lymphoid cells Abstract

All protocols are subject to individual parameters of each assay.