Anti rfc4 rabbit polyclonal antibody

Total cellular proteins were extracted from tissues or cells, separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (Pall, New York, USA). Membranes were blocked with 5% nonfat milk in 1% Tween-PBS (PBST) and then probed overnight with anti-RFC4 rabbit polyclonal antibody (1:1000, Epitomics, Burlingame, CA, USA) or anti-ACTB antibody (1:1000, Proteintech, Chicago, IL, USA). After three washing steps of 10 min in PBST, membranes were incubated with species-appropriate fluorescently-conjugated secondary antibodies (1:10000 in PBST, LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. The immunoreactive signals were detected using the two-color fluorescent western blotting Odyssey infrared imaging system (LI-COR Biosciences).

The TMAs were deparaffinized in xylene and rehydrated with graded ethanol. Sections were then heated in antigen retrieval solution (EDTA, pH 9.0) for 20 min and incubated with 3% H₂O₂ for 10 min. The sections were then incubated with anti-RFC4 rabbit polyclonal antibody (1:800, Epitomics) at 4°C overnight. The sections were then treated with the secondary antibody for 15 min at room temperature and stained with 3, 3′-diaminobenzidine until brown granules appeared in the membrane, cytoplasm, or nucleus (Dako, EnvisionSystem/DAB-chromogen, Glostrup, Denmark). The sections were counterstained with hematoxylin for 2 min at room temperature. A negative control was employed by exchanging the specific primary antibody with non-immune serum immunoglobulins at a 1:200 dilution.