3 3 diaminobenzidine (dab)

The formalin-fixed paraffin-embedded (FFPE) lung cancer tissue array was obtained (Biomax, Derwood, MD, USA, catalog no. BC041115d). EBERs (EBER1 and EBER2) ISH was performed using the HistoSonda EBER XISH Probes kit (American MasterTech, Lodi, CA, USA). Briefly, the FFPE tissue sections were deparaffinized, rehydrated in a graded solution of xylene and alcohol, and deproteinized with proteinase K. Samples were incubated with a digoxigenin EBER probe and washed with deionized water and 1× PBS. They were incubated first with anti-digoxin antibody and anti-mouse horse peroxidase antibody and subsequently with 3,3′-diaminobenzidine (Biocare Medical, Pacheco, CA, USA), counterstained with hematoxylin (Sigma, St. Louis, MO, USA), and washed again with 1× PBS. Slides were then dehydrated in a graded solution of xylene and alcohol and subsequently sealed with the VectaMount permanent mounting medium (Vector Laboratories, Burlingame, CA, USA). Slides were scanned with an Aperio CS2 digital pathology scanner, and images were obtained with Aperio ImageScope software (version 12.3.2.8013, Leica, Buffalo Grove, IL, USA) with 40× magnification.

Tumors isolated from NSG mice were sectioned and verified by P.H., a board-certified pathologist. Sections of formalin-fixed, paraffin-embedded tissues were immunostained with antibody to human RAD51 (ThermoFisher scientific cat# PA5-27195) followed by anti-Rabbit secondary antibody (Biocare Medical MACH2 Rabbit HRP-Polymer; catalog # RHRP520#) and staining with 3,3'-diaminobenzidine (Biocare Medical). Gill”s Hematoxylin III and Lithium Blue (Poly Scientific) were used as counterstains. Nuclear staining was quantified using ImageJ. Percent RAD51 positive nuclei were quantified in 3 random fields per tumor (each field containing approximately 150–200 cells), and the average percent (mean ± std deviation) RAD51 positive nuclei calculated for each tumor.

Sections of formalin-fixed paraffin-embedded (FFPE) de-identified human tissues from HGSOC (9 patients), endometroid (9 patients), normal ovary (6 subjects) and normal fimbriae (obtained from 5 of the 9 HGSOC patients for which normal fimbriae were available) were obtained from the biorepository of UCHC (IRB IE-08-310-1). Tissues were immunostained with antibodies to human SFXN4 (Sigma cat# HPA020872) followed by anti-rabbit secondary antibody (Biocare Medical MACH2 Rabbit HRP-Polymer; catalog # RHRP520#) and staining with 3,3'-diaminobenzidine (Biocare Medical). Gill’s Hematoxylin III and Lithium Blue (Poly Scientific) were used as counterstains. Images of three to four random fields per slide (each field containing approximately 150–200 cells) were quantified as described⁴⁵ ; each dot in the figure represents the average value obtained for an individual subject. Data is presented as relative intensity units; thus, only samples analyzed in the same staining session are compared (i.e., normal fimbriae vs HGSOC; normal OSE vs HGSOC; and normal OSE vs endometrioid were each stained and quantified separately). A board-certified pathologist (P.H.) provided histomorphologic confirmation of the ovarian cancer subtypes and normal adnexal tissues and their corresponding immunostains that were quantified in this analysis.

Sections from paraffin-embedded tissues were incubated with a goat anti-human B7-H3 antibody (1:200, R&D Systems, #AF1027), a rabbit anti-human KIF15 antibody (1:2000, Proteintech, #55407-1-AP) or a rabbit anti-human Ki67 antibody (1:500, Abcam, ab15580) overnight at 4 °C. This step was followed by staining (45 min at room temperature) with the corresponding HRP-labeled rabbit anti-goat secondary antibody or goat anti-rabbit secondary antibody (Invitrogen). Next, the sections were visualized by staining with 3,3’-diaminobenzidine (Biocare Medical, CA, USA) and counterstaining with hematoxylin (Sigma). The numbers of Ki67-positive cells and total cells were analyzed using a microscope (Leica, Buffalo Grove, USA).
All sections were then reviewed blindly by two experienced pathologists (Dr. Cao and Dr. Zhan). The scoring criteria for B7-H3 and KIF15 immunostaining were based on clinical data and adopted the semiquantitative immunoreactive score (IRS) system^{17 (link)}. Briefly, category A (intensity of immunostaining) was scored using the following criteria: 0, negative; 1, weak; 2, moderate; and 3, strong. Category B (percentage of immunoreactive cells) was scored using the following criteria: 1 (0–25%); 2 (26–50%); 3 (51–75%); and 4 (76–100%). Final scores were calculated by multiplying the scores of categories A and B in the same section; the scores ranged from 0 to 12.