Figure 3.

Application of stripping buffer instead of HIER results in improved quality of the septuple immunofluorescence staining technique. Opal multiplex immunofluorescence staining of human skin using HIER with Tris-EDTA buffer (A) or β-mercaptoethanol-containing stripping buffer (B) for antibody removal. Representative immunofluorescence images showing CD45 (in cyan), CD3 (in purple), CD8 (in green), Melan-A (in orange), cytokeratin (in yellow), mast cell tryptase (in red) and DAPI (in blue). Arrow heads indicate cells that detached during HIER treatment and subsequently randomly stuck to the slide. Bars equal 100 µm. Abbreviation: HIER, heat-induced epitope retrieval; EDTA, ethylenediaminetetraacetic acid; DAPI, 4′,6-diamidino-2-phenylindole.