Mouse anti glp 1

Proteins from cell lysates were denatured with SDS buffer and boiled for 5 min. Samples were run on an SDS-PAGE gel, transferred onto polyvinylidene difluoride (PVDF) membrane, and blocked with 5% milk. Then, the membranes were probed with the following primary antibodies overnight at 4°C: mouse anti-GLP-1 (1:1000, Abcam, MA, United States) and rabbit anti-β-actin (1:1000, Santa Cruz, CA, United States). They were incubated with goat anti-rabbit-HRP IgG (1:10,000, Santa Cruz, CA, United States) and goat anti-mouse-HRP IgG (1:10000, Santa Cruz, CA, United States) for 1 h at room temperature. Immunoreactive bands were detected by chemiluminescence techniques after washing three times and then visualized on Kodak Omat X-ray film. The intensity of the specific bands was calculated using ImageJ software version 1.44p. GLP-1 alterations reflect cellular production of GLP-1 in the intestine.

Transverse sections of rat kidneys (6 μm) were fixed with cold acetone for 10 min. The sections were then washed with phosphate-buffered saline (PBS), permeabilized for 30 min with 0.25% Tx-100 in PBS, and blocked for 40 min with 10% normal goat serum or with 5% BSA, prior to incubation overnight at 4°C with primary antibodies rabbit anti-CD26, mouse anti-GLP-1 and rabbit anti-GLP-1R from Abcam, and goat anti-IL-1β and rabbit anti-TNF-α from R&D Systems (Minneapolis, MN, USA). The sections were rinsed with PBS and then incubated with 4′,6-diamidino-2-phenylindole (DAPI) and secondary fluorescent antibodies for 1 h at room temperature. After washing, samples were imaged using a confocal microscope (LSM 710, Carl Zeiss, Gottingen, Germany).