4.6. Immunohistochemistry for p53 protein (I)

Immunohistochemistry was used to study the p53 protein and its sub-cellular localization. The skin sections were fixed in neutral 10% formalin, embedded in paraffin and cut into 5 m sections. The sections were mounted on slides coated with poly-L-lysin, deparaffinized in xylene and dehydrated in graded ethanol. The sections were then incubated in 0.1% hydrogen peroxide in absolute methanol to block endogenous peroxidases and in 20% foetal calf serum in phosphate-buffered saline to block non-specific binding. The avidin-biotin complex method was used. The sections were incubated with polyclonal antibody CM5 for 12 hours. Sections treated in a similar manner without CM5 were used as negative controls. The sections were then incubated with secondary anti-rabbit immunoglobulin and the avidin-biotin complex (Dako A/S, Denmark). The brown colour for positive staining was developed with diaminobenzidine, and the samples were lightly counterstained with haematoxylin. The nucleus thus stains brown in a positive and blue in a negative case. The percentages of immunopositive keratinocytes were counted in five high-power (40 × objective) fields including at least 500 keratinocytes each.