Pms2 clone a16 4

Primary cell lines MSI status was determined using a panel of five markers (BAT25, BAT26, D2S123, D5S346 and BAT40) for Polymerase Chain Reaction (PCR) as well as standard IHC techniques. Briefly, evaluation of MMR protein expression was performed on 5-µm sections of the tissue or cell blocks using antibodies to MLH1 (clone G168-15, 1:75 dilution, Biocare Medical, Concord, CA), PMS2 (clone A16-4, 1:300 dilution, Biocare Medical), MSH6 (clone BC/44, 1:100 dilution, Biocare Medical) and MSH2 (clone FE11, 1:100 dilution, Biocare Medical). Detection was performed using the MACH 3 Mouse HRP-Polymer Detection Kit (Biocare Medical). Chromogenic detection was achieved with diamniobenzidine (Dako) and sections were counterstained with hematoxylin (Dako). Results were evaluated by a board certified gynecologic pathologist experienced in MMR protein immunohistochemical interpretation. Staining of any tumor nuclei was interpreted as positive, with expression by lymphocytes and/or stromal cells considered a positive internal control. Tumors were classified as MSI-low if they had one unstable marker, MSI-high if they had two or more unstable markers and MSI-negative/MSS if all markers were stable by PCR.