Ab8068

The expressions of Vimentin and Keratin in rat peritoneal mesothelial cells were validated by immunofluorescence method. Briefly, rat peritoneal mesothelial cell slides were prepared, fixed with 4% paraformaldehyde for 30 min at room temperature, treated with 0.2% Triton X-100 for 60 min at room temperature for permeabilization, and blocked with 4% BSA solution for 40 min at room temperature. Subsequently, rat peritoneal mesothelial cell slides were washed with PBS and incubated with the anti-Vimentin (#ab92547; Abcam; 1:500) or anti-Keratin (#ab8068; Abcam; 1:500) for 1.5 h at room temperature and 95% humidity, followed by incubation with fluorescent-conjugated secondary antibodies for 2 h at room temperature. To evaluate the subcellular localization of PPARγ and GLUT1 proteins, rat peritoneal mesothelial cells were treated as above-mentioned using anti-PPARγ gamma (#ab272718; Abcam; 1:500) and anti-GLUT1 (#12939; CST; 1:500) antibodies, which were finally observed using laser confocal microscope.

Tumour and normal tissues were embedded in paraffin and cut into 4 µm-thick sections. The sections were de-paraffinised using xylene and rehydrated using a gradient concentration of ethanol; the antigen was extracted using microwaves. Subsequently, the sections were incubated with anti-KRT5 (ab8068; Abcam) at 4°C overnight and then with a secondary antibody (ab288151; Abcam) at room temperature for 30 min. DAB plus substrate (D7679, Sigma-Aldrich) was added and the sections were incubated for 10 min. The results were visualised using a microplate reader.