Heat-Assisted Enhanced Peroxidase One-Step Method

MICROWAVE HEAT-ASSISTED ENHANCED PEROXIDASE ONE-STEP METHOD 

The enhanced peroxidase one-step (EPOS) method is considered superior to standard ABC technique in that the former is more sensitive than the latter. It is known that the Ki-67 antibody can only be used on fresh or frozen tissues, whereas the monoclonal antibody MIB-1, developed against a part of the Ki-67 antigen molecule, can be used on sections of formalin-fixed and paraffin-embedded tissues using antigen retrieval. Recently, EPOS Ki-67 antibodies were developed which consist of antibody molecules and horseradish peroxidase bound covalently to dextran (Bisgaad et al., 1993). This method has been applied for localizing PCNA and Ki-67 antigens (Tsutsumi et al., 1995). 

Surgically removed tissues are fixed with 4% buffered formalin and embedded in paraffin (Turner et al, 1999). Sections (4 p.m thick) on slides are deparaffinized, rehydrated, and then treated with 3% H202 to block endogenous peroxidase. They are placed in 0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave oven. An EPOS rabbit antihuman Ki-67 antibody is applied as supplied (DAKO) it is ready to be used without any dilution. Color development is accomplished with metal-enhanced DAB for 15 min, followed by light counterstaining with hematoxylin. Quantification of Ki-67 antibody-labeled 

MICROWAVE HEAT-ASSISTED IMMUNOSTAINING OF CELL SMEARS 

Consistent and reliable immunostaining of cytological preparations can be obtained 

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