Rabbit anti mouse β catenin

The preparation of kidney tissue homogenates and western blotting analysis were performed as previously described.[20 (link)] For the detection of TGF-β1 and β-catenin, blots were incubated with rabbit anti-mouse TGF-β1(1:5000; Abcam, Hong Kong) and rabbit anti-mouse β-catenin (1:10,000; Abcam, Hong Kong) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000; Abcam, Hong Kong). All western blot results were normalized to β-actin.

B16-BL6 cells were co-cultured with Tregs for 72 h, and then 5 × 10⁵ cells were injected subcutaneously into the right abdomen of 6- to 7-week-old male C57BL/6 mice. Tumor tissues were harvested from mice at 15 days after cancer cell injection and fixed in 10% formalin, processed for paraffin embedding, and cut into 5 μm-thick sections. Tumor sections were immunostained with rabbit anti-mouse TGF-β, rabbit anti-mouse α-SMA, rabbit anti-mouse β-catenin, rabbit anti-snail/slug, or rabbit anti-vimentin purchased from abcam. After incubating with primary Ab at 4 °C overnight, sections were incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG (cat. no. A11008, Invitrogen, Carlsbad, CA) or Alexa Fluor 568-labeled goat anti-rabbit IgG (cat. no. A11001, Invitrogen) at room temperature for 1 h. For counterstaining, the samples were incubated with 4,6-diamidino-2-phenylindole (Sigma, St. Louis, MO, USA). Slides were viewed under a confocal laser-scanning microscope (LSM510, Carl Zeiss MicroImaging, Thornwood, NY, USA).