Rat anti ly6g

For tissue specimens, mice were transcardially perfused with phosphate-buffered saline (PBS). Organs were reduced to single-cell suspensions by 2 digestions with collagenase type 2 (Sigma) and collagenase/dispase (Roche). Preparations were passed through a 40-μm cell strainer (Becton Dickenson) and washed. The resulting single cells were collected, blocked in 5% FBS in PBS, centrifuged and then stained with the following antibodies: rat anti F4/80-APC (eBioscience), rat anti TIE2-PE (eBioscience) and rat anti-CD11b-PE-Cy7C conjugated. Cells were labeled using 7-amino-actinomyocin D before analysis to detect necrotic cells.
Blood collected from each animal before and 3 days after STZ/CTRL treatment and then at weekly intervals during SILD/vehicle treatment was anti-coagulated with EDTA at room temperature. Ammonium Chloride solution was used for 10 minutes at RT to allow red blood cell lysis. Cells were stained with rat anti-CD11b conjugated in PE-Cy7 (eBioscience) and rat anti-Ly-6G conjugated in APC (eBioscience) and were then centrifuged at 1200 RPM for 10 min at 4°C. Typical antibody-specific dilutions were used according to supplier datasheet. All samples were collected by a CyAn ADP cytometer (DAKO). Biexponential analysis was performed using Summit V4.3 software.

Expression of the target ligand/receptor was detected using anti-rat, anti-rabbit, or anti-goat horseradish peroxidase (HRP)–diaminobenzidine (DAB) staining kits (R&D Systems), depending on the primary antibody, and according to the manufacturer's instructions. Briefly, sections were rehydrated, and endogenous peroxidase activity was blocked. Antigen unmasking was achieved by incubating the sections in prewarmed trypsin–EDTA (0.1%) in phosphate buffered saline (PBS) for 30 minutes at 37°C. Following the blocking steps, the sections were incubated overnight with 4 μg/ml of rat anti–Ly-6G (Invitrogen), goat anti–matrix metalloproteinase 9 (anti–MMP-9; Santa Cruz Biotechnology), rabbit anti-CXCL1 (Clontech), goat biotinylated anti–DR-3 (R&D Systems), or isotype controls diluted in PBS followed by biotinylated secondary antibody, according to the manufacturers' instructions. Sections were counterstained with hematoxylin, dehydrated, and mounted in DPX. Positive staining was visualized using a streptavidin–HRP conjugate and DAB chromogen that stained positive areas brown. Images were captured using a digital camera (Olympus N457 or Canon EOS 100D), and the proportion of brown pixels within a particular area was measured using Adobe Photoshop CS3.5. Five randomly selected areas were used to generate scores for each sample.