Eslidemanager database

YY1 IHC staining was conducted using a rabbit anti-YY1 antibody (Atlas Antibodies #HPA001119) and the Bond fully automated slide staining system (Leica Microsystems). Slides were dewaxed in Bond Dewax solution (AR9222) and hydrated in Bond Wash solution (AR9590). Heat-induced antigen retrieval was performed for 30 min at 100°C in Bond-Epitope Retrieval solution 1 pH-6.0 (AR9961), followed with a 5-min Bond peroxide blocking step (DS9800). After pretreatment, slides were incubated for 30mins with primary antibody YY1(1:150) followed with Vector Immpress HRP anti-rabbit IgG (MP-7401-15). Chromogenic detection of all antibodies was performed using the Bond Intense R Detection kit (DS9263). Stained slides were dehydrated and covered with a slip. Positive and negative controls (no primary antibody) were included for each run. Stained slides were digitally scanned at 20x magnification using Aperio ScanScope-XT (Aperio Technologies, Vista, CA, USA). The images were uploaded to the Aperio eSlideManager database (Leica Biosystems Inc; eSlideManager version 12.3.3.7075) and scored by the Translational Pathology Laboratory at UNC. 

Organs were fixed in 10% formalin and paraffin embedded. Tissue sections were stained with H&E according to the specifications of Leica's H&E protocol using SelecTech Define MX-aq, SelecTech Blue Buffer 8, SelecTech Hematoxylin 560 MX, SelecTech Alcoholic Eosin 515. Immunohistochemical analysis of CD45R (protein tyrosine phosphatase receptor type C; lymphocyte common antigen) was performed as described [19] (link) using rat monoclonal anti-CD45R (clone RA3-6B2, 1:800 dilution). Anti-rat IgG and peroxidase detection system reagents (DAB chromogen) were purchased from Abcam, Vector Laboratories, and BioGenex, respectively. Slides were scanned into the eSlide Manager database (version 12.3.2.5030) with the Aperio ScanScope CS (Leica, Biosystems, Inc.). Using Aperio macros, the number of cells stained with DAB chromogen and the total number of cells in tumor sections were quantified. Statistical analysis was carried out using a Student's T-test.