Bond epitope retrieval solution 1 er1

Formalin fixed paraffin embedded (FFPE) tissue sections were de-paraffinized and endogenous peroxidase was inactivated. Antigen retrieval was accomplished by heat/pressure cook using the Bond Epitope Retrieval Solution 1 (ER1) at 99–100°C for 30 minutes (Leica Microsystems). Following retrieval, the sections were incubated sequentially with the primary antibody for 25 minutes, post-primary for 15 minutes and polymer for 25 minutes ending with colorimetric development with diaminobenzidine (DAB) for 10 minutes (Bond Polymer Refine Detection; Leica Microsystems). Antibodies used were: ERG (1:100 Epitomics #28051) and ERG status was assessed as previously described^{32 (link)}. Briefly, subjective evaluation of ERG protein expression determined for each tumor core using a four-tier grading system: negative (0), weakly positive (1+), moderately positive (2+), and strongly positive (3+)in target cells above background (see Supplemental Table.

Immunohistochemistry (IHC) was performed using the anti-HNF4G antibody HPA005438 (MilliporeSigma, Billerica, MA, USA) at a 1:50 dilution, on a Bond III automated immunostainer (Leica Microsystems, IL, USA). Formalin-fixed paraffin-embedded (FFPE) tissue sections were de-paraffinized and endogenous peroxidase was inactivated. Antigen retrieval was performed using the Bond Epitope Retrieval Solution 1 (ER1) at 99–100°C for 60 minutes (Leica Microsystems). Sections were then incubated sequentially with the primary antibody overnight, post-primary for 15 minutes and polymer for 25 minutes, followed by a 10 minute colorimetric development with diaminobenzidine (DAB) (Bond Polymer Refine Detection; Leica Microsystems). FFPE material from 22Rv1 prostate cancer cell line xenografts with known levels of HNF4G expression were used as controls.
IHC was performed on tissue microarrays composed of representative cases of benign human prostate tissue, hormone naïve prostatic carcinoma (HNPCa), castration-resistant prostatic adenocarcinoma (CRPC). Staining was evaluated by a pathologist with experience in genitourinary pathology (JC). Nuclear HNF4G expression was considered positive if at least 10% of nuclei in a given tissue type showed brown staining. Nuclear staining intensity was scored following a three-tiered system (negative=0, weak=1, medium=2 and strong=3).