Avidin biotin blocking

The immunofluorescent staining was performed at Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems). The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems) and antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 min with Background Buster solution (Innovex), followed by avidin-biotin blocking (Ventana Medical Systems) for 8 min. Slides were incubated with anti-pErk1/2 (Cell Signaling, cat# 4370, 1 μg/ml) and anti-Mac1 (abcam, cat#ab133357, 1 μg/ml) for 5 hr, followed by 60 min incubation with biotinylated goat anti-rabbit (Vector Labs, cat# PK6101, 5.75 μg/ml) at 1:200 dilution. The detection was performed with Streptavidin-HRP D (part of DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen, cat# T20922, for pErk1/2) and Tyramide Alexa Fluor 568 (Invitrogen, cat#T20914, for Mac1) prepared according to the manufacturer’s protocol with predetermined dilutions. After staining slides were counterstained with DAPI (Sigma Aldrich, cat# D9542, 5 μg/ml) for 10 min and coverslipped with Mowiol.

The immunofluorescence was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems). The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 min with Background Buster solution (Innovex), followed by avidin-biotin blocking for 8 min (Ventana Medical Systems). Multiplex immunofluorescence stainings were performed as previously described^{53 (link)} (see Supplementary Note). Stained slides were digitized using Pannoramic Flash 250 (3DHistech, Hungary) using 20x/0.8NA objective. Regions of interest were drawn on the scanned images using Pannoramic Viewer (3DHistech, Hungary) and exported into tiff images. ImageJ/FIJI was used to segment DAPI-stained nuclei and count the cells with positive signal. Ki67 was quantified according to the recommendations for breast cancer^{54 (link)}, where the percentage of positively stained nuclei is quantified among the total number of malignant cells as previously described¹³ .

Tissue microarrays (purchased from US Biolab) containing a total of 126 prostate tumor specimens from 66 patients with localized and metastastic disease were stained for β-catenin expression by immunofluorescence through the Molecular Cytology Core Facility at MSKCC using a Discovery XT processor (Ventana Medical Systems). Briefly, tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems) and antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 minutes with Background Buster solution (Innovex), followed by avidin-biotin blocking for 8 minutes (Ventana Medical Systems). Sections were incubated with a β-catenin antibody (8814; Cell Signaling) for 5 hours, followed by a 60-minute incubation with biotinylated goat anti-rabbit IgG (PK6101; Vector labs) at a 1:200 dilution. Detection was performed with Streptavidin-HRP D (part of DABMap kit, Ventana Medical Systems), followed by incubation with Tyramide Alexa 488 (B40953; Invitrogen) prepared according to the manufacturer’s instructions. After staining, slides were counterstained with DAPI (D9542; Sigma Aldrich) for 10 min and coverslipped with Mowiol. Tissues were then scored on a 0–3 scale for β-catenin expression, with scores of 0 and 1 as “negative” and 2 and 3 as “positive” for β-catenin.

Progression biopsies and collection of patient samples were conducted under appropriate Institutional Review Board/Privacy Board protocols and waivers (protocols 06–107, 12–245, 14–019). Participating patients signed written informed consent for these biospecimen protocols. This study was conducted in accordance with ethical guidelines in the Declaration of Helsinki. Immunohistochemistry was performed by the Molecular Cytology Core Facility in MSKCC. The tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 minutes with Background Buster solution (Innovex), followed by avidin-biotin blocking for 8 minutes (Ventana Medical Systems). Sections were incubated with anti-ARAF (Santa Cruz sc-408) or anti-BRAF (Sigma HPA001328) antibodies for 5 hours, followed by 60 minutes incubation with biotinylated horse anti- rabbit (Vector Labs, cat# PK6101) at 1:200 dilution. The detection was performed with DAB detection kit (Ventana Medical Systems) according to manufacturer instruction. Slides were counterstained with hematoxylin and coverslipped with Permount (Fisher Scientific).