Heat-Assisted Evaluation of Global DNA Hypomethylation

MICROWAVE HEAT-ASSISTED EVALUATION OF GLOBAL DNA HYPOMETHYLATION 

Qualitative and quantitative differences can be observed and measured between the normal and malignant part of each tumor specimen. Morphologically altered nuclei display densely labeled spots within faintly labeled areas, whereas normal nuclei are darker and uniformly stained. 

Malignant lesions and normal tissue biopsies (colon) are fixed with formalin and embedded in paraffin (Hernandez-Blazquez et al., 2000). Paraffin sections (4 p,m thick) are deparaffinized, rehydrated, and rinsed in PBS. They are placed in 0.1 mM citrate buffer (pH 3.4), heated for 10 min in a microwave oven at full power (720 W), and washed in PBS. The slides are immersed in 2N HC1 for 2 hr at 37°C and then rinsed in PBS. The sections are covered with 100 p,l ofhybridoma supernatant containing anti-5-MeCyd monoclonal antibody (5 (xg/ml) and incubated for 1 hr at room temperature with a biotinylated goat antimouse secondary antibody diluted 1 200 in PBS containing 0.1% BSA. 

The sections are rinsed in PBS and then treated for 30 min with streptavidin-peroxidase, diluted 1 100 with PBS-BSA. They are rinsed in PBS and treated with DAB-H202 for 5 min. The PBS used for rinsing contains 0.1% Tween 20. 

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