Beclin 1 primary antibody

Immunohistochemical methods were utilized to assess autophagy-related Beclin-1 levels in three tissue microarrays (TMAs) containing EAC tissues which differed by tumor grade and stage for a subset of biopsies (n =54). Specifically, TMAs BS02051, ES208, and ES804 were purchased from US Biomax, Inc. (Rockville, MD). A total of 115 EAC biopsies were evaluated from 51 patients. Additionally, 59 normal squamous esophageal biopsies from 21 patients were assessed for Beclin-1 immunoreactivity. Methods included a 5 min antigen retrieval in sodium citrate, a 3% peroxidase block, 1 h incubation with primary antibody Beclin-1 (Anti-rabbit, NB500-249, 1:1000, Novus Biologicals, Littleton, CO) and detection with Envision +System-HRP/DAB (Dako, Carpenteria, CA). Each Beclin-1 stained biopsy was scored on an intensity scale spanning from “0” or no staining to “+3” for intense staining comprising greater than 30% of the tumor or epithelium in the case of normal tissues. Beclin-1 loss was characterized by an intensity score in the 0–1 range; whereas scores in the 2–3 range were considered positive for Beclin-1. Staining was confirmed using a second Beclin-1 primary antibody (Santa Cruz Biotechnology, Dallas, TX, #sc-11427; 1:100).