Ultraview universal diaminobenzidine dab detection kit

IHC was performed on 4-μm-thick formalin-fixed, paraffin-embedded (FFPE) tissue sections using anti-AR rabbit monoclonal primary antibody (prediluted, pH 9, catalog no. 760-4605, Roche-Ventana) and anti-ACE2 rabbit monoclonal primary antibody (1:100, pH 9, catalog no. GTX01160, GeneTex). IHC was carried out on the Benchmark XT automated slide staining system (Roche-Ventana Medical Systems) using the UltraView Universal diaminobenzidine (DAB) detection kit (catalog no. 760-500, Roche-Ventana) and Hematoxylin II (catalog no. 790-2208, Roche-Ventana) for counterstain. Dual IHC was performed consecutively, and signals were developed using the Universal DAB detection kit and the Discovery purple kit (catalog no. 760-229, Roche-Ventana). Staining was evaluated under 100× and 200× magnification using a bright-field microscope. See SI Appendix for scoring details.

Deparaffinized and rehydrated sections from the lung and heart were used for immunohistochemistry. Immunostaining was done with BenchMark Ultra IHC/ISH System fully automated instrument (Roche, Basel, Switzerland), using the ultraView Universal diaminobenzidine (DAB) Detection Kit (760–500, Roche-Ventana, Tucson, AZ 85755, USA), in accordance with the standard protocols supplied by the manufacturer. All antibodies were ready-to-use monoclonal antibodies (Ventana, Roche Diagnostics, Basel, Switzerland); antibodies directed against CD3 (Ventana, 2GV6) CD4 (Ventana, SP35), CD8 (Ventana SP57), CD20 (Ventana, LZ6), CD68 (Ventana KP-1), and CD45 (Ventana, LCA) were used. Negative control stainings were performed by omitting the primary antibodies.
All cases were independently analyzed by two pathologists without knowledge of the clinical diagnosis. The extent of immunoreactivity was assessed by using the same microscope at a magnification of 40×. The number of positive cells was quantified and expressed as cells/mm².

For immunohistochemical analysis, 4 μm TMA-sections were automatically pre-treated using ULTRA Cell Condition Solution 1, pH 8.5 (Ventana Medical Systems, Tucson, AZ, USA) for heat induced epitope retrieval, and stained with the monoclonal antibodies CONFIRM anti-ER (SP1) and anti-PR (1E2) in a Ventana BenchMark stainer (Ventana Medical Systems). The antibody-antigen complex was visualized with UltraView Universal diaminobenzidine (DAB) Detection kit (Ventana Medical Systems).
Stained slides were digitalized at 20x magnification using the automated scanning system Hamamatsu, NanoZoomer, 13239-01, Hamamatsu Photonics, Sunayama-cho, Naka-ku, Hamamatsu City, Shizuoka, Japan). The immunohistochemical staining was manually annotated by three independent observers (GA, KJ and JEL) who were blinded to the clinical data and outcome of the patients, and with the two latter being board-certified pathologists, using the NDP.view2 viewer software version 3.2.12 (Hamamatsu Photonics). Expression of ER and PR was annotated as the number of tumor-associated stromal cells with nuclear staining. Conflicting annotations were jointly re-evaluated in order to reach consensus. The intensity of staining was not evaluated.