Immunohistochemical staining for AR-V7 was done on serial 4 μm breast tissue sections as described previously [62 (link)] using the AR-V7 EPR15656 antibody (1:200), biotinylated anti-rabbit antibody (1:400, DAKO Corp., Carpinteria, CA), streptavidin-horseradish peroxidase complex (1:500, DAKO), and diaminobenzidine tetrahydrochloride. To analyze 22Rv1 cells (Supplementary Figure S4 ), a standard cytospin protocol was utilized [63 (link)] followed by immunohistochemistry as above.
Preparation of formalin-fixed, paraffin-embedded tissue sections for immunofluorescence was done as described previously [64 (link)]. For all antigens, retrieval was performed in 600 mL of 10 mM Tris base and 1 mM Na-EDTA (pH 9.0) by heating in a 1100W microwave at full power for 5 min and subsequently heating at 50% power for an additional 5 min. Primary antibodies used for immunofluorescence were AR-441 (M3562, 1:50, DAKO) and AR-V7 (EPR15656, 1:400). Primary antibodies were detected using secondary antibodies conjugated to either Alexa-Fluor 488 (A11029; Life Technologies) or Alex-Fluor 568 (A11036; Life Technologies). Images were acquired sequentially on a Zeiss 700 confocal microscope with a pinhole aperture of 2 airy units.
Preparation of formalin-fixed, paraffin-embedded tissue sections for immunofluorescence was done as described previously [64 (link)]. For all antigens, retrieval was performed in 600 mL of 10 mM Tris base and 1 mM Na-EDTA (pH 9.0) by heating in a 1100W microwave at full power for 5 min and subsequently heating at 50% power for an additional 5 min. Primary antibodies used for immunofluorescence were AR-441 (M3562, 1:50, DAKO) and AR-V7 (EPR15656, 1:400). Primary antibodies were detected using secondary antibodies conjugated to either Alexa-Fluor 488 (A11029; Life Technologies) or Alex-Fluor 568 (A11036; Life Technologies). Images were acquired sequentially on a Zeiss 700 confocal microscope with a pinhole aperture of 2 airy units.