Mouse anti ki67 antibody

Mouse mammary tissues were extracted, fixed in formalin and embedded in paraffin before sectioning at 5 μm. Haemotoxylin and eosin staining was carried out according to standard protocols. For immunohistochemical analysis of Ki67, slides were immersed in 0.5 % hydrogen peroxide in methanol for 10 minutes to inhibit endogenous peroxidase activity, followed by antigen retrieval by boiling slides in 0.1 M sodium citrate buffer under pressure. Slides were blocked for 20 minutes in 5 % normal rabbit serum in TBS/0.1 % Tween to prevent non-specific antibody binding, and then incubated overnight at 4 °C with mouse anti-Ki67 antibody (Vector Labs) according to the manufacturer’s instructions. Specific antibody binding was detected using the EnVision Dual Link System (Vector Labs), followed by incubation with diaminobenzidine (DAB) substrate (Dako). Sections were counterstained with haemotoxylin, dehydrated and mounted.

Tumor samples were formaldehyde-fixed and paraffin-embedded. Sections were dewaxed and rehydrated following standard procedures. When required, heat-induced epitope retrieval was performed by incubating sections in 10 mM sodium citrate buffer (pH6.0) heated at 95°C for 20 min. Sections were cooled down at room temperature and were then stained using appropriate Vectastain ABC kits (Vector Laboratories, Burlingame, CA). Primary antibodies used were: mouse anti-human vimentin antibody (Leica, Davie, FL), mouse anti-Ki67 antibody (Vector Laboratories), rabbit anti-cleaved caspase 3 (Cell Signaling, Danvers, MA), rabbit anti-CD31 (PECAM) antibody (Abcam, Cambridge, MA), rabbit anti-LTBP3 (Santa Cruz Biotechnology, Dallas, TX), Sections were subsequently counterstained with methyl green or hematoxylin. Hematoxylin and eosin and Masson’s trichrome stainings were performed following standard procedures.