Immpact novared hrp substrate

Details regarding antibodies are provided in Supplemental Table 2. Biological samples, including patient tissue, were provided by the MD Anderson Cancer Center. Critical commercial assays included the Chromium Single Cell 3′ Reagent Kit (10x Genomics) and the ImmPACT NovaRED HRP substrate (Vector Laboratories). C57BL/6J mice were obtained from The Jackson Laboratory (strain 000664).

To assess angiogenesis, tumors were stained for new vessel formation (CD31) and smooth muscle cell recruitment (αSMA). Tumors were sectioned and baked at 58°C for 1 hour. Antigen unmasking was performed by heat-induced epitope retrieval using 0.05% citraconic anhydride solution (PH 7.4) for 45 minutes at 98°C. Samples were blocked with 1% BSA for 30 minutes at room temperature then incubated with antibodies against CD31 (1:300, Abcam, RRID: AB_726362) or αSMA (1:2000, Abcam, RRID: AB_2223021) overnight at 4°C. The sections were visualized with ImmPACT NovaRED (HRP) Substrate (Vector Laboratories) and counterstained with hematoxylin Gill Method 1 (Fisher Scientific). Slides were scanned at 20X with a Hamamatsu Photonics Nanozoomer Slide Scanner in the Virtual Pathology Core. Visiopharm digital pathology analysis software (Version 2020.08, Visiopharm, RRID: SCR_021711) and custom-designed applications were used to quantify the percent of positive immunostained areas. A region of interest was drawn around the tissue, the area of the positive staining was identified and measured within the region of interest, and the ratios of the positive staining area to the total area were calculated.

Sections of 5-μm of paraffin-embedded tissues were deparaffinized in two changes of xylene for 5 min each and then rehydrated in distilled water using graded alcohols. Antigen retrieval was done by steaming the slides for 20 min then cooling for 20 min in EDTA buffer (1 mM EDTA, 0.05% Tween 20, pH 8) for AID and PRMT1. Endogenous peroxidase was blocked with a 0.3% hydrogen peroxide solution for 10 min. Endogenous biotin was blocked for 15 min with the blocking buffer provided with the Avidin/Biotin System (#SP2001; Vector Laboratories). For protein block, we used 10% normal goat serum and 1% BSA for 60 min at room temperature. Sections were incubated with anti-AID (1:50, rat Mab mAID-2 eBioscience), anti-PRMT1 (1:100) overnight at 4°C. Biotin-conjugated secondary antibodies were mouse anti–rabbit IgG (1:200; Vector Laboratories) to detect anti-PRMT1; mouse anti–rat IgG (1:200; Vector Laboratories) to detect anti-AID. Biotinylated reagents were detected with Vectastain ABC kit (PK-6100; Vector Laboratories). Peroxidase activity was developed using ImmPACT NovaRED HRP substrate (Vector Laboratories). Sections were counterstained with hematoxylin (cat. #MHS32-1L; Sigma-Aldrich) for 1 min prior to dehydrating and mounting for imaging on a bright field microscope.