Figure 2

Schematic demonstration of the three HIF1α antibodies used in this study and their epitopes (Figs. 2a and b). Ab1 is a polyclonal antibody raised against N-domain of wild type HIF1α and does not recognize HIF1α1.2 due to it's different N-terminal part (Fig. 2a). Ab2 and Ab3 are monoclonal and polyclonal antibodies, respectively, with epitopes in the common parts of HIF1α and HIF1α1.2 (Fig.2a and b). Immunohistochemical analysis performed on thin adjacent sections of NE-differentiated prostate cancer using the HIF1α antibodies Ab1 (Fig. 2c), Ab 2 (Fig. 2d), Ab 3 (Fig. 2e) and HIF1β antibody (Fig. 2f). Immunopositivity was detected for HIF1α with Ab2 and Ab3 while HIF1α Ab1 produced no detectable staining. HIF1β was also positive in adjacent section (Fig. 2f). Double staining of chromogranin A and androgen receptor antigens on adjacent sections (Fig. 2g) showed immunopositivity for chromogranin A (Fast red). Androgen receptor antibody (DAB, brown) produced no staining. Immunostaining of benign prostate tissue with HIF1α Ab3 showed immunopositivity in NE-like cells of benign prostate tissue (Fig. 2h). In addition, HIF1β antibody recognized benign NE-like cells in benign prostate hyperplasia (Fig. 2i). Double staining with HIF1α Ab3 and HIF1β (Fig. 2j) shows co-localization of the two proteins in NE-like cells of benign prostate hyperplasia (HIF1α Ab3 red stain, HIF1β brown stain). Panels a, b, c, d, e, f and g: 40× objective. Panels h, i and j: 60× objective.