Sc 9966

Fixed bones and tumors [4% formalin] were partially decalcified in 14% EDTA for 3 days, followed by paraffin embedding, sectioning then staining with hematoxylin (Sigma)) and eosin (Sigma) (H&E). For Immunohistochemistry, paraffin-embedded tissues were deparaffinized, followed by antigen retrieval with 25 mM citrate buffer (PH 6) in a pressure cooker. Then, the sections were left to cool for 25 min, followed by blocking of the endogenous peroxidase with 3% H₂O₂ (Sigma) for 15 min. To reduce non-specific binding of the primary antibody, tissues were blocked with blocking solution (CAS Block) (Invitrogen), followed by incubation with the primary antibody overnight at 4 °C. Sections were washed with TBST, followed by incubation with secondary anti-mouse immunoglobulin antibody for 30 min (Nichirei Biosciences). The reaction was then performed using a DAB peroxidase kit (Vector Laboratories, SK-4100), followed by hematoxylin (Bar Naor, Cat# BN4537N) staining for 40 s as a counterstain. IHC stains were manually counted, at least five pictures were taken for each slide, and the average was calculated for each slide. The antibodies used were mouse monoclonal MCM7 (141.2) [sc-9966-Santa Cruz 1:200].

The antibodies used for Western blot (WB) are listed, and their dilutions are shown below. The monoclonal antibody against GAPDH (sc-365062, 1:500 for WB) and the monoclonal against GFP (sc-9966, 1:1000 for WB) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); monoclonal antibodies against MCMV IE1 and M112-113 proteins were provided by Dr. Stipan Jonjic (Medicine University of Rijeka, Rijieka, Croatia; 1:200 for WB) [9 (link),18 (link)]; the monoclonal antibody against M25 was a generous gift from Dr. Shanley (University of Connecticut., Stamford, CT, USA; 1:250 for WB) [19 (link),20 (link)].