Alexa fluor 568 conjugated goat anti rabbit igg secondary antibody

The cultured cells were washed one time with cold PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Following washing three times with PBS, the cells were permeabilized with 0.2% Triton X-100 in PBS buffer for 20 minutes at room temperature. Following washing three times with 0.1% Tween20 in PBS, the cells were blocked with 0.1% Tween20 and 10% goat serum, with 1% BSA in PBS buffer for 1 hour at room temperature. The cells were incubated with the anti-rabbit primary antibodies of β-catenin (Abcam, Cambridge, UK), Axin1 (Novus Biologicals, Centennial, USA), Axin2 (Novus Biologicals) and phosphorylated LRP6 (Biorbyt Ltd., Cambridge, UK) over night at 4°C. The cells were washed three times with 0.1% Tween20 in PBS. The cells were incubated with Alexa Fluor^® 568 conjugated goat anti-rabbit IgG secondary antibody (Invitrogen, Carlsbad, USA) for 45 minutes at room temperature. To visualize the nuclei, the cells were double-stained with 4’, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, USA). The cells were viewed with a Keyence BZ 800 epifluorescence microscope, which was equipped with a digital camera (CFI 60, Nikon Corporation, Tokyo, Japan). All immunofluorescence images were obtained with identical exposure settings.

Pancreatic tissue samples were fixed with 4% paraformaldehyde at 4°C and then embedded in paraffin. Serial sections of 4-μm thickness were cut off from each paraffin block at 200-μm intervals, and five sections were randomly selected per mouse and deparaffinized. The sections were incubated with guinea pig anti-insulin polyclonal antibody (1:100; ab7842; Abcam, Cambridge, UK), or rabbit anti-Ki67 monoclonal antibody (1:100; GTX16667; GeneTex, Irvine, CA, USA) for 48 h at 4°C. The sections were then treated with Alexa Fluor 488-conjugated goat anti-guinea pig IgG secondary antibody (1:500; A11073; Invitrogen) or Alexa Fluor 568-conjugated goat anti-rabbit IgG secondary antibody (1:500; A11036; Invitrogen) for 1.5 h at room temperature. The total areas of insulin-positive cells and the number of islets were analyzed, and Ki67-positive β-cells were quantitatively assessed as a percentage of the total number of β-cells. All fluorescently stained sections were examined with a BZ9000 fluorescent microscope system (Keyence, Osaka, Japan).