Tissuefaxs plus

Tumor tissues were dissected, embedded in OCT compound (4583, Sakura), and frozen immediately at −80°C. Serial 8-μm sections were collected from the cryostat. After fixation in 4% paraformaldehyde for 20 min and blocking in 10% donkey serum in 1× PBS at room temperature for 1 hour, the sections were permeabilized in PBS containing 0.1% Triton X-100. The sections were then incubated overnight at 4°C with the following primary antibodies: anti–E-cad (1:200; 610181, BD Biosciences), anti-WNT5A (1:50; ab235966, Abcam), anti-NRG1 (1:100; ab191139, Abcam), anti–α-SMA (1:200; A5228, Sigma-Aldrich), anti-ERBB3 (1:100; 12708, Cell Signaling Technology), anti-PDGFRα (1:200; 3174, Cell Signaling Technology), and anti-FZD6 (1:100; NBP1-89702, Novus Biologicals). Tissue sections were washed three times with PBS and incubated with the appropriate Alexa Fluor–conjugated secondary antibodies [donkey anti-rabbit Alexa Fluor 488 (1:500; A-21206, Invitrogen), donkey anti-mouse Alexa Fluor 568 (1:500; A10037, Invitrogen), donkey anti-mouse Alexa Fluor 568 (1:500; A10042, Invitrogen), or donkey anti-mouse Alexa Fluor 488 (1:500; A-21202, Invitrogen)] for 1 hour at room temperature, after which they were incubated with DAPI for 15 min. Images were obtained on a TissueGnostics imaging system (TissueFAXS Plus, Austria).

The TissueFAXS Plus (Tissue Gnostics, Vienna, Austria) automated microscopy workstation, which contains an eight‐slide ultra‐precise motorized stage and utilizes a Zeiss Z2 Axioimager microscope (Zeiss, Oberkocken, Germany), was used for automated image acquisition. First, a DAPI preview image is captured with a ×10 objective to allow for appropriate orientation. Next, the TMA spots are identified and images are captured with a ×40 oil objective using the DAPI, GFP, Cy3, and Cy5 filters. An autofocus algorithm in the DAPI filter and the extended focus parameter by capturing three steps above and below (step size = 0.8 μm) was utilized. An entire TMA with 400 spots can be imaged in ~14 h, which is faster than other current imaging modalities [13 (link)]. For image analysis, a separate high‐performance workstation with the TissueQuest software module to analyze the fluorescent images with precise nuclear segmentation was used [12 (link), 14 (link)]. A region of interest is set (e.g. stroma or cancer) and processed for nuclear segmentation. If required, exclusion regions were set to exclude benign prostate glands.