Cy3 affinipure goat anti mouse igg

Cells on sterile slips were fixed in 4% parafomaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Next, the slips were incubated with E-cadherin (1:50, #20874-1-AP, Proteintech) or Vimentin (1:100, #60330-1-Ig, Proteintech) antibody overnight at 4 °C. Subsequently, the slips were incubated with FITC labeled goat anti-rabbit IgG (1:1000, #ab6717, Abcam) or Cy3-AffiniPure Goat Anti-Mouse IgG (1:500, #115-165-003, Jackson) for 1 h. DAPI was used for nuclear staining. Finally, fluorescence was imaged under the fluorescent microscope (IX71, Olympus, Japan).

Rat monoclonal anti-BrdU (#OBT0030; AbDSerotec, Raleigh, NC), rabbit polyclonal anti-cytoskeletal actin (β-actin) (#A300-491A; Bethyl Laboratories, Inc., Montgomery, TX) and mouse monoclonal anti-Ki67 (NCL-Ki67-MM1; Novocastra/Leica Microsystems, Inc., New Castle, UK), rabbit polyclonal to p85 fragment of PARP (#G734A, Promega, Madison, WI) and cyclin B (BD #61029; BD Biosciences, San Jose, CA) were procured from their respective manufacturers. Rabbit polyclonal to cleaved caspase-3 (#9661), p21^Cip1/WAF1 (#CS2947), Cdc25C (#CS4648), p-Cdc25C (#CS9528), Cdc2 (#CS9112) and p-Cdc2 (#CS9111) were purchased from Cell Signaling Technology, Inc., Danvers, MA. Antibodies for p53 (#SC-6243), p27^kip21 (#SC528), p19 (#SC-71810), cyclin E (#SC481), Cdk2 (#SC6248) and Cdk4 (#SC23896) were all from Santa Cruz Biotechnology Inc., CA. Peroxidase-conjugated secondary (H&L chain-specific) antibodies for Western blots, goat anti-mouse IgG (#401253) and goat anti-rabbit IgG (#401315) were purchased from Calbiochem-Novabiochem Corp., CA. Secondary antibodies for immunocytochemical analyses, Cy3-AffiniPure donkey anti-rat IgG (#712-165-153) and Cy3-AffiniPure goat anti-mouse IgG (#115-165-003) were procured from Jackson Immuno Research Laboratories, Inc., West Grove, PA.

The assay was performed as previously described (Houlihan et al., (link)). Briefly, paraffin‐embedded kidney slides were deparaffinized and rehydrated with xylene, ethanol and water sequentially. Antigens were unmasked by incubating slides in an Antigen unmasking solution (H3300, Vector lab) and boiling for 20 min. Non‐specific binding was blocked by incubation with 0.05% Triton‐X 100 and 5% normal goat serum (005‐000‐121, Jackson ImmunoResearch) in PBS at room temperature for 1 h. Slides were then incubated with a rabbit antibody against cleaved caspase‐3 (9661, Cell Signaling Technology) and mouse antibody against β‐catenin (610153, BD Transduction Laboratories) and Hoechst 33342 for staining nuclei (Molecular Probes) at 4°C overnight. An Alexa Fluor 488 AffiniPure Goat ant‐rabbit IgG (111‐545‐144, Jackson ImmunoResearch) and a Cy3 AffiniPure Goat anti‐mouse IgG (115‐165‐146, Jackson ImmunoResearch) were used to recognize the respective anti‐cleaved caspase‐3 and β‐catenin antibodies.