Scn400 scanning system

Immunohistochemical stains were conducted at the Vancouver Prostate Centre using a Ventana autostainer model Discover XT (Ventana Medical System) with enzyme labeled biotin streptavidin system and solvent resistant DAB Map kit using 1/50 concentration of rabbit polyclonal IL-33 antibody (HPA024426; Sigma-Aldrich). Staining was performed on 342 prostate cancer specimens obtained from the Vancouver Prostate Centre. The H&E stained slides were reviewed and the desired areas were marked on them and their correspondent paraffin blocks. Five tissue microarrays (TMAs) were manually constructed (Beecher Instruments) by punching duplicate cores of 1mm for each sample. Stained slides were digitalized with the SL801 autoloader and Leica SCN400 scanning system (Leica Microsystems) at magnification equivalent to ×20. Representative cores (clearly positive, clearly negative and mixed positive/negative) were manually identified by an experienced pathologist (L Fazli) and a four-point scale was assigned as follows: 0 represents no staining in any tumour cells, 1 represents a faint or focal, or questionably present stain, 2 and 3 represents a stain of convincing intensity in a majority of cells. For comparisons, a score of 0 or 1 was considered low IL-33 expression.

Freshly cut tissue microarray (TMA) sections were analyzed for immunoexpression using Ventana Discovery Ultra autostainer (Ventana Medical Systems, Tucson, AZ). In brief, tissue sections were incubated in Tris-EDTA buffer (CC1) at 37 °C to retrieve antigenicity, followed by incubation with respective primary antibodies at room temperature or 37 °C for 60–120 min. For primary antibodies, mouse monoclonal antibodies against CD8 (Leica, NCL-L-CD8-4B11, 1:100), CK5/cytokeratin 5 (Abcam, ab17130, 1:100), BAP1 (SantaCruz, clone C4, sc-28383, 1:50), rabbit monoclonal antibody against CD3 (Abcam, ab16669, 1:100), and rabbit polyclonal antibodies against CALB2/calretinin (LifeSpan BioSciences, LS-B4220, 1:20 dilution) were used. Bound primary antibodies were incubated with Ventana Ultra HRP kit or Ventana universal secondary antibody and visualized using Ventana ChromoMap or DAB Map detection kit, respectively. All stained slides were digitalized with the SL801 autoloader and Leica SCN400 scanning system (Leica Microsystems; Concord, Ontario, Canada) at magnification equivalent to × 20. The images were subsequently stored in the SlidePath digital imaging hub (DIH; Leica Microsystems) of the Vancouver Prostate Centre. Representative tissue cores were manually identified by two pathologists.

All stained slides were digitalized with the SL801 autoloader and Leica SCN400 scanning system (Leica Microsystems; Concord, Ontario, Canada) at magnification equivalent to ×40. The images were subsequently stored in the SlidePath digital imaging hub (DIH; Leica Microsystems) of the Vancouver Prostate Centre. The scoring method used was based on assigning a value on a four-point scale to each immunostain. Descriptively, 0 represents no staining by any tumor cells (negative), 1 represents a faint or focal, questionably present stain (weak), 2 represents a stain of convincing intensity in a minority of cells (moderate), and 3 a stain of convincing intensity in a majority of cells (strong).

Prostate cancer specimens were obtained from the Vancouver Prostate Centre Tissue Bank according to institutional guidelines. Specimens were examined by Hematoxylin and eosin staining and desired areas were marked on the paraffin blocks. TMAs were manually constructed (Beecher Instruments) by punching duplicate cores (1 mm) for each sample (Table S4). TMAs were stained with antibodies specific to POU4F1 (1:320 in Ventana Discovery antibody diluent; Polyclonal; Catalogue number: AB5945; Merck) or TUBB3 (1:100 in Ventana Discovery antibody diluent; Clone: TUJ1; #801201, Biolegend) using the Ventana Discover XT™ autostainer (Ventana Medical Systems) and scanned with a Leica SCN400 scanning system (Leica Microsystems). Due to the non-homogeneous nature of prostate cancer, scoring was performed manually by an experienced pathologist (Dr. Ladan Fazli). The scoring consisted of a four point scale: 0 = no staining of tumour cells; 1 = faint/focal or questionable staining; 2 = staining of convincing intensity in the majority of the tumour cells; and 3 = staining of strong intensity in the majority of tumour cells. Details on number of patients and staining categories can be found in Table S3.

This study was done on the total of 19 radical prostatectomy specimens obtained from Vancouver Prostate Centre Tissue Bank. The study protocol was approved by University of British Columbia (UBC) Clinical Research Ethics Board (CREB) and Vancouver Coast Hospital Research Institute Research (VCHRI) Research Ethics Board (REB). All patients gave informed consent as approved by UBC CREB and VCHRI REB. Immunohistochemical staining was conducted by Ventana autostainer model Discover XT™ (Ventana Medical System, Tuscan, Arizona) with enzyme‐labeled biotin–streptavidin system and solvent‐resistant DAB Map kit using 1/400 concentration of mouse monoclonal antibody against Cdc25C and 1/800 concentration of goat polyclonal antibody against CLU. All stained slides were digitalized with the SL801 autoloader and Leica SCN400 scanning system (Leica Microsystems, Concord, Ontario, Canada) at magnification equivalent to ×20 and subsequently stored in the SlidePath digital imaging hub (DIH; Leica Microsystems) of the Vancouver Prostate Centre. For each biomarker, representative cores (clearly positive, clearly negative, and mixed positive/negative) were manually identified by a pathologist and values on a four‐point scale were assigned.