Method of melanin bleaching in MIB1-Ki67 immunostaining of pigmented lesions: A quantitative evaluation in malignant melanomas

The effect of melanin bleaching on the immunoreactivity of the MIB1-Ki67 an tigen in pigmented melanocytic lesions was investigated. Eight paired non-p igmented and heavily pigmented malignant melanomas (6 primary melanomas and 2 secondary melanomas) were selected. Avidin-biotin immunoperoxidase compl ex (ABC) and microwave antigen retrieval were used in immunostaining. Secti ons were incubated with 10% H2O2 for 24 h before immunostaining with primar y antibody MIB1, or after the completion of immunostaining. Non-bleached co ntrols were obtained by conducting the identical staining but omitting the bleaching procedure. In all heavily pigmented lesions bleached by 10% H2O2 before or after immunostaining, the melanin was bleached effectively and MI B1-positively stained cells were clearly seen. Cell counting in the non-pig mented group found that there were no significant differences in the percen tage of MIB1-positive melanoma cells (%MIB1) between non-bleached controls and those sections which had been bleached by 10% H2O2 either before or aft er the immunostaining. The results suggest that hydrogen peroxide can effec tively bleach melanin in pigmented melanocytic lesions without significantl y affecting MIB1-Ki67 immunolabelling.