Goat anti rabbit igg fluor

Fixed kidney tissues were embedded in paraffin. For both IHC and IF, sections were deparaffinized, rehydrated, washed in PBS containing 0.1% Tween 20 (PBST) and blocked in 10% normal goat serum. The following primary antibodies were applied to sections and incubated at 4°C overnight: β-Catenin, cyclin D1, KI-67 and pGSK3β (Cell Signaling Technology, Inc., MA, USA), GSKα (Sigma Aldrich, MO, USA), GSK3β (Santa Cruz Biotechnology, Inc. TX, USA), PCNA (Dako, CA, USA), DBA and LTA (Vector Laboratories, CA, USA). For IHC, slides were blocked with Avidin/Biotin (Invitrogen), and then biotinylated goat anti-rabbit IgG or anti-mouse IgG (Invitrogen, NY, USA) secondary antibodies were applied, followed by incubation with Streptavidin HRP conjugate (Invitrogen, NY, USA). Finally slides were developed with DAB (Vector Laboratories) and counterstained with Harris Haematoxylin, dehydrated, and mounted with Permount (Fisher Scientific). For IF, after incubation with primary antibody, goat anti-Rabbit IgG fluor, Goat anti-mouse IgG Texas red (Invitrogen, NY, USA), or Goat anti-chicken IgG Alexa 555 (Sigma Aldrich, MO,USA) secondary antibodies were applied, and following incubation, washed, stained with DAPI and mounted with Flour-G (Invitrogen, NY, USA). All images were captured using a Nikon 80i microscope in KUMC imaging center.

Fixed and paraffin tissue sections were processed as described before (31 (link), 32 (link)). FoxM1 and Ki-67 (cat # 12202S Cell Signaling Technology, Inc., MA, USA) antibodies were used for immunofluorescence (IF) co-staining. For immunohistochemistry, secondary antibodies were applied, followed by incubation with Streptavidin-HRP conjugate (Invitrogen, NY, USA) and slides were developed with DAB (Vector Laboratories) and counterstained with Harris Haematoxylin, dehydrated, and mounted with Permount (Fisher Scientific, NJ, USA). For IF, goat anti-Rabbit IgG fluor and Goat anti-mouse IgG Texas Red (Invitrogen, NY, USA), secondary antibodies were applied, stained with DAPI, and mounted with Flour-G (Invitrogen, NY, USA). All images were captured using a Nikon 80i upright microscope (Tokyo, Japan) in the KUMC imaging center.