Mouse monoclonal anti bcl2 primary antibody

Mammary tissues specimens were fixed in 10% buffered neutral formalin, washed, dehydrated, cleared, embedded in paraffin, casted and finally sectioned. Deparaffinization was done using xylene. About 3% H₂O₂ was added for 10 min to inactivate the peroxidases. Then antigen retrieval was performed by heating at 121°C in 10 mM citrate buffer for 30 min then blocking was done for 20 min in 5% normal serum. After that sections were incubated with mouse monoclonal anti-Bcl2 primary antibody (sc-7382; Santa Cruz Biotechnology, Inc., Dallas, TX) in PBS overnight at 4°C. After washing with PBS, sections were incubated with a goat anti-rabbit IgG biotin-conjugated secondary antibody (1:2,000; sc 2040; Santa Cruz Biotechnology, Inc., Dallas, TX). After incubation with horseradish peroxidase-labeled streptavidin, antibody binding was visualized using diaminobenzidine, and sections were counterstained with hematoxylin [22 (link)].

HPLC grade reference standards 3,4,5,-trihydroxybenzoic acid (gallic acid ethyl ester, purity 98%) was purchased from Acros Organics, Fischer Scientific; and 3,3′,4′,5,6-pentahydroxy flavones (quercetin dihydrate, purity 98%) was purchased from Fluka Biochemika. Urea and thiourea were purchased from Hi-Media Laboratories, India. 2,3,7,8-Tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione (ellagic acid), dithiothreitol (DTT), acrylamide, bis-acrylamide, ammonium persulfate, tetramethylethylenediamine (TEMED), sodium dodecyl sulphate (SDS), tris-HCL, phenylmethanesulfonylfluoride (PMSF), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), tween 20, and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate were purchased from Sigma-Aldrich, USA. Polyvinylidene fluoride (PVDF) membranes were purchased from Millipore (India) Pvt., Ltd., India. Mouse monoclonal anti-Bcl-2 primary antibody, anti-beta-actin primary antibody, rabbit polyclonal anti-Bax primary antibody, HRP conjugated anti-mouse IgG secondary antibody, HRP conjugated anti-rabbit IgG secondary antibody, and nonfat dry milk were purchased from Santa Cruz Biotechnology, CA. Methanol, glacial acetic acid, formaldehyde, hematoxylin and eosin stains, and other analytical chemicals and reagents used for HPLC and phytochemical analyses were purchased from Merck Pvt., Ltd., India.