Ab180717

Tissue sections were paraffin-embedded and immunostained with an antibody against USF1 (ab180717, Abcam). The tissue specimens were routine dewaxed. EDTA (pH 8.0) was used for microwave repair. After that, the endogenous peroxidase was blocked by adding 3% hydrogen peroxide in distilled water after natural cooling, incubated at room temperature for 10 min, and rinsed three times with phosphate buffer (PBS) for 2 min. The specimens were covered with 1:100 diluted MAGE-A3 monoclonal antibody, incubated for 1 h at 37°C, and rinsed three times with PBS for 5 min each. HRP-labeled secondary antibody was added to cover the specimen, incubated for 20 min at 37°C, and rinsed with PBS. The HRP-labeled secondary antibody was added to cover the specimen and was incubated for 20 min at 37°C. Three times rinsing with PBS followed for 5 min each. Diaminobezidin (DAB) was added to control the color development under the microscope. Rinsing thoroughly with tap water, re-stained with hematoxylin, dehydrated, and observed under the microscope after the tablets were sealed.

MCs were inoculated and cultured on a 35 mm glass base dish (IWAKI 3910-035, Tokyo, Japan) in DMEM with 10% FBS and normal glucose. After serum starvation in DMEM with 0.5% FBS for 24 h, cells were incubated in DMEM with 0.5% FBS and normal glucose or high glucose for 20 h. Then, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized in PBS with 0.25% Triton-X for 15 min. After blocking with 10% albumin for 20 min, cells were incubated with rabbit anti-USF1 antibody (ab180717, Abcam, Cambridge, UK) diluted to 1:100 for 1 h. After washing with PBS, cells were incubated with Alexa-594 Goat anti-Rabbit IgG (A11072, Invitrogen, Waltham, MA, USA) as the second antibody for 30 min. Nuclei in the cells were stained with Hoechst 33342 (H1399, Invitrogen) diluted 1:1000.