Purified Mouse Anti-Human MLH1 Clone G168-728 (RUO)

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Briefly, 2 μm sections of representative samples were cut from paraffin-embedded, invasively growing colorectal carcinoma specimens. Surrounding normal colonic mucosa served as an internal control. Sections were deparaffinized twice with xylene and rehydrated in five graded alcohol baths. Antigen retrieval by heating was performed in a pressure cooker for 15 min in EDTA buffer, pH 8.0. This was followed by incubation for 10 min with 3% H2O2 to block endogenous peroxidase activity. Sections were washed with 1x PBS (Gibco, USA) before and in between incubation steps. Primary MLH1 antibody (clone G168-728, 1:500 dilution) or primary SPTAN1 antibody (clone C-11, 1:250 dilution) were diluted in PBS containing 1% BSA. Sections were incubated with one primary antibody at 4°C overnight, followed by application of the EnVision System mouse (K4000, Agilent, USA), which employs the enzyme horseradish peroxidase and the chromogen 3,3’-diaminobenzidine (DAB). Samples were treated with the peroxidase reagent DAB for 10 min, diluted to 1 drop of DAB chromogen per ml of DAB substrate buffer (K3467, Agilent, USA). Sections were counterstained using Gill’s hematoxylin solution. Immunohistochemical staining was examined using a Keyence microscope (Model BZ-9000, KEYENCE Co., Osaka, Japan). Negative controls were processed in parallel to exclude non-specific staining.

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