Sl801 autoloader

Following formalin fixation, tissues were embedded in paraffin. Sections (4um) were then prepared and mounted on slides for staining. Antigen retrival was performed using citrate buffer (pH 6.0) and steamed at high power for 17 min. Immunohistochemical staining of anti-NTN-1 (rabbit monoclonal [EPR5428] ab126729 Abcam Cambridge, UK) was achieved using the Ventana autostainer Discover XT (Ventana Medical Systems Oro Valley, AZ). Ultra-map DAB anti-Rb detection kit (760-151 Roche Basel, CH) was used as secondary antibody. Stained slides were digitalized with the SL801 autoloader and Leica SCN400 scanning system (Leica Microsystems Wetzlar, DE).

Immunohistochemical staining was performed using freshly cut 5 micron sections from each site shipped to Stanford University and a commercial antibody for MUC1 (1:50 dilution; SC-7313, Santa Cruz Biotechnology) [20 (link)]. The digital image documentation of all stained slides was performed using the Leica SCN400 scanning system with the SL801 autoloader (Leica Microsystems; Concord, Ontario, Canada) at magnification equivalent to 40x. The images were transferred into the SlidePath digital imaging hub (DIH; Leica Microsystems). In parallel, separate TMA sections were stained with hematoxylin and eosin (H & E) and high molecular weight keratins (HMWK, 34bE12, Dako); these sections were scored for the presence of cancer in each core on the TMA as described previously [21 (link)–26 (link)]. A single pathologist (LF) scored MUC1 protein staining only in cores in which cancer was present as determined using the H & E and HMWK.
The immunohistochemical staining intensity for MUC1 was defined as absent, weak (faint cytoplasmic staining of scattered cells), moderate (intermediate or heterogeneous cytoplasmic staining in tumor cells), or strong (dense cytoplasmic staining of nearly all tumor cells) as shown in Fig 1.

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).