Clone g168 728

Immunohistochemical staining for Mlh1, Msh2, Msh6 and Pms2 proteins was performed in lymphoma cells from the single Msh2^wt mouse that displayed an MSI lymphoma following Aza treatment. Briefly, 4 μm sections were incubated with monoclonal antibodies against MLH1 (1/20 dilution, clone G168–728, Pharmingen, San Diego, CA), MSH2 (1/25 dilution, clone FE11, Calbiochem, Oncogene Research Products, Cambridge, MA), MSH6 (1/25 dilution, clone 44, Becton Dickinson, Lexington, NC) and PMS2 (1/20 dilution, clone A16–4, BD PharMingen, Le Pont de Claix, France).

IHC was performed on 4-μm-thick sections of the fresh-frozen specimens with a BenchMark XT automated immunostainer (Ventana Medical Systems Inc., Tucson, AZ). Primary monoclonal antibodies against MLH1 (clone G168-728, diluted 1:250, BD PharMingen, San Diego, CA), MSH2 (clone FE11, diluted 1:50, Oncogene Research Products, La Jolla, CA), MSH6 (clone 44, ready to use, Ventana Medical Systems Inc.), and PMS2 (clone A16-4, diluted 1:200, BD PharMingen) were used. For external controls, non-neoplastic colonic mucosa and colorectal tumors known to be deficient in MLH1, MSH2, MSH6, and PMS2 were employed. Evidence of expression of each protein was defined by nuclear IHC reactivity. The absence of MMR protein expression was defined by the total absence of nuclear staining. Even very weak staining was considered proficient.