Cat hematoxylin

Tumor tissues were stained with the B7-H3 mAb overnight at 4 °C (clone 376.96, 1:1000 dilution, final concentration 1 μg/mL)^{43 (link)}, and then stained with HRP polymer conjugated goat anti-mouse secondary Ab (Dako, code K4000, 1:8 dilution) at 25 °C for 1.5 h. Slides were developed using DAB chromogen (Cell signaling), counterstained with CAT hematoxylin (Biocare medical), dehydrated in ethanol, and cleared in xylene (Fisher chemical). Cover slips were added using a histological mounting medium (Fisher, toluene solution). Stained TMA slides were digitally imaged at ×20 objective using the AperioScanScope XT (Leica). TMA slides were de-arrayed to visualize individual cores and each core was visually inspected. Folded tissues were excluded from the analysis using a negative pen, and all other artifacts were automatically excluded with the Aperio Genie software. The B7-H3 positive score was measured using Aperio membrane v9 (cell quantification) algorithm. Percentage of positive cells obtained with this algorithm at each intensity level (negative, low, medium, high) were used to calculate the H-Score using the formula: H-Score = (% at 1+) × 1+(% at 2+) × 2+(% at 3+) × 3. The Aperio color deconvolution v9 algorithm with the Genie classifier was also applied to calculate the area and intensity of the positive stain and generate a Score (0–300).

Sections were cut, deparaffinised, and rehydrated as described above. Antigen retrieval was performed for 30 s at 123°C in Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) using a pressure cooker followed by a cool-down period of 20 min. Reduction of peroxidases was accomplished by incubating for 8 min in Peroxidazed 1 (BioCare Medical, Concord, CA). Slides were sequentially blocked in Avidin-Biotin Blocking Kit (BioCare Medical), Background Sniper (BioCare Medical) and Rodent Block M (BioCare Medical) for 15 min, respectively. Biotinylated Hyaluronic Acid Binding Protein (Millipore, Billerica, MA) was added at a 1:800 dilution and incubated for 1 h at room temperature followed by a 10 min incubation with 4+ Streptavidin HRP Label (BioCare Medical). Staining was performed with Betazoid DABKit (BioCare Medical) for 5 min followed by counterstaining with CAT Hematoxylin (BioCare Medical) for 1 min.

All tissues collected were stained with CAT hematoxylin (Biocare Medical, CA, USA; catalog no. CATHE) and Edgar Degas Eosin-Y (Biocare Medical, CA, USA; catalog no. HTE) also known as H&E staining. We used picro-sirius red stain kit (Abcam, Cambridge, MA, US; catalog no. ab150681) to evaluate tissue fibrosis. Histopathological evaluation of the colon, kidney, spleen, heart, gonadal fat, and liver sections of all animals of Control and treated with Low Quercetin, Medium Quercetin, and High Quercetin groups was performed blindly by a trained graduate student (P.C.) and a certified pathologist (I.C.). The specimens were evaluated for findings of inflammation, dysplasia, and fibrosis.