Icam 1 antibody

Micro-vascular endothelial cells were treated with ice-cold lysis buffer containing 10 mmol/l PMSF (pH, 7.5). The lysates were then subjected to SDS-PAGE and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. PVDF membranes were incubated overnight at 4ºC with one of the following primary antibodies, including rabbit anti-mouse ELAM-1 antibody, VCAM-1 antibody, ICAM-1 antibody, IKBα antibody, p-IKBα antibody, and GAPDH antibody (all antibodies were purchased from Abcam Biotechnology, Cambridge, Massachusetts, USA). Subsequently, the PVDF membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Beyotime Biotechnology Inc.), at room temperature for 1 hour. Bound antibodies in the above PVDF membranes were developed with chemiluminescent substrate (ECL, Beyotime Biotechnology Inc.), according to the instruction of manufacturer. The immunoreactive bands were imaged and scanned with gel imaging system (Tannon-4200, Tannon, Shang, China).

ICAM-1 antibody was obtained commercially (Abcam, Cambridge, Mass). Donor grafts were embedded in paraffin, and sections were deparaffinized in xylene followed by treatment in 100% ethanol, 95% ethanol, 80% ethanol, and 50% ethanol (5 minutes, 2 times each). Tissue sections were then rinsed with water and 13 phosphate-buffered saline buffer. Antigen retrieval was performed by boiling in antigen unmasking solution (Vector Laboratories, Inc, Burlingame, Calif) for 30 minutes followed by cooling the samples to room temperature.
Tissue sections were washed and permeabilized and then stained for ICAM-1 (1:200). Tissue sections from each experimental time points including 18 hours of CI (n ¼ 3 for control and vildagliptin groups), 7 days (n ¼ 5 for control and vildagliptin groups), and 14 days (n ¼ 3 for control and vildagliptin groups) after Tx were used and the fluorescent signal from 5 regions within each sample were evaluated. Images were acquired with a Zeiss Meta 510 laser scanning confocal and multiphoton microscope with Zeiss 2007 software (Zeiss, Inc, Thornwood, NY). All immunofluorescence analyses were performed at the Institute for Environmental Medicine and Department of Physiology, University of Pennsylvania Perelman School of Medicine (Philadelphia, Pa).