Ab6884

For immunofluorescence microscopy, cells were plated on cover slides and fixed in 4% paraformaldehyde for 15 min. The fixed cells were permeabilized on 0.5% Triton X-100 for 30 min, and blocked with 10% goat serum for another 30 min, then incubated with Vimentin (Santa Cruzs), Ki67 (Proteintech) overnight at 4 °C. After extensively washing with PBS, secondary antibody, the donkey anti-goat IgG conjugated with Alexa Flour 594 (ThermoFisher, A-11058), was applied to the slides for 1 h. After three times washing, cell nuclei were stained with DAPI (Sigma) for 5 min. The slides were photographed by confocal microscopy.
For IHC, paraffin-fixed tissues were deparaffinized, rehydrated and treated with 0.01 M sodium citrate for antigen retrieval. Endogenous peroxidase activity was blocked by 0.3% (v/v) hydrogen peroxide and then incubated with 0.3% Triton X-100 for 15 min. After blocking with 10 % goat serum for 1 h. The tissues were incubated with primary antibodies at 4 °C overnight. After extensive washing with PBS, tissues were incubated with secondary antibodies (Abcam, ab6884) for 1 h. Diaminobenzidine hydrogen peroxide was used to show staining signaling, then the sections were counterstained with hematoxylin.

For IHC, paraformaldehyde-fixed tissues were washed with PBS for three times. After blocking with 10% donkey serum for 1 h at room temperature, tissues were incubated with primary antibodies at 4 °C overnight. After three times washing, tissues were incubated with secondary antibodies (Abcam, ab6884) for 1 h at room temperature. Diaminobenzidine-hydrogen peroxide (Sigma) was the chromogen, and the counterstaining was carried out with 0.5% hematoxylin. The intensities of IHC staining was classified into the following grades:(-, no staining; +, 10%; ++, 10%-50%; +++, >50%). For IF, cells were seeded on coverslips in a 24-well plate and immunofluorescence staining was conducted as previously described 25 (link). The primary antibody was goat anti-KLB. The secondary antibody was donkey anti-goat IgG conjugated with Alexa Fluor 594 (ThermoFisher, A-11058). Cell nuclei were stained with DAPI (Sigma) for 5 min at room temperature. Stained cells were photographed and quantified under an immunofluorescence microscope (Leica DFC420C). Two experienced pathologists scored the KLB staining independently, and the final score was the score average from both pathologists.

Biopsy cores (18G, 17mm) were taken from the prostate peripheral zone following removal by robotic radical prostatectomy or radical cystoprostatectomy performed to treat PCa or bladder cancer, respectively (see Supplemental Table S1). Sextants harboring PCa at the transrectal biopsy and a nodule were targeted. The biopsies were washed with PBS and cultured for 24 hours in eMEM-10%FBS supplemented with 10nM DHT and antibiotics and the PCA3-3STA (10⁷ ivp) adenovirus under gentle shaking. The biopsies were then transferred on a Gelfoam soaked into fresh media for another 48h, after which time luciferin was added and the luminescence activity was imaged by means of Xenogen IVIS [31 (link)]. The biopsies were then fixed in 10% formalin and embedded in paraffin. Five-μm sections were analyzed with either H&E staining or immuno-histochemical staining (IHC). The luciferase IHC was performed with the IDetect Super Stain System HRP (IDST1007, ID Laboratories, London, ON, Canada) with anti-luciferase (1:7000, 70R12141, Fitzgerald, Acton, MA, USA) as the primary antibody and Donkey anti-Goat antibody (1:500, Ab6884, Abcam, Toronto, ON, Canada) as the secondary antibody. The IHC with the PSA, androgen receptor (AR), Ki67 or cocktail of AMACR/p63 antibodies were performed by the clinical pathology facility at the CHU de Quebec Hospital using a standard clinical protocol.