Figure 4

The effects of different buffered bleaching methods on immunogenicity. We performed immunohistochemistry for Ki-67 and HMB45 after the usage of the bleaching technique with heavily melanin-pigmented melanoma FFPE tissues. (a) Representative immunohistochemical images of Ki-67 and HMB45 after melanin bleaching (scale bar: 100 μm). (b) Immunogenicity measured using mean percentage of Ki-67 and mean histoscore of HMB45 indicated that Tris/Tricine/SDS and Tris-HCl (pH 10) yielded the highest signals for nuclear staining (all P<0.001) and cytoplasmic staining (all P<0.001). We also performed immunohistochemistry for Ki-67 and HMB45 before and after the usage of the bleaching technique with melanoma tumor specimen without melanin pigment. (c) Relative expressional signal of both Ki-67 and HMB45 was normalized to that of unbleached melanoma tissue specimen. The antigenicity of Ki-67 and HMB45 was relatively well-preserved after the bleaching methodology using Tris-HCl (pH 10) buffer. NB, non-bleaching. (d) Representative immunohistochemical images of Ki-67 and HMB45 before and after melanin bleaching (scale bar: 100 μm).