Procyte dx

Intravital microscopy was performed using LRP1^ctrl and LRP1^cKO mice as well as C57Bl/6 mice 2 h after intrascrotal injection of TNFα (500 ng per mouse) as previously described (Weckbach et al., 2014 (link)). For experiments with C57Bl/6 mice, anti-N-MK Ab (Cellmid) or the matching isotype control Ab (clone MOPC-21; 400101; BioLegend) were applied i.p. 12 h before the experiment. Briefly, after cannulation of the carotid artery, the cremaster muscle was surgically prepared as reported previously (Weckbach et al., 2014 (link)). Intravital microscopy was conducted using an upright microscope (Axiotech Vario, Zeiss; and DM6 FS, Leica) at 37°C and recorded using a digital camera (AxioCam HSm, Zeiss; and Zyla sCMOS, Andor Technology). Leukocyte counts were obtained from whole blood (ProCyte Dx; IDEXX Laboratories). Cells attached >30 s were defined as adherent. Diameter of postcapillary venules ranged from 20 to 40 µm. Centerline red blood cell velocities in microvessels were analyzed using fluorescent microspheres (0.5 µm diameter; Polysciences). Blood flow was calculated from the length of at least three microspheres measured in a snapshot image with a defined exposure time and converted offline to mean blood flow velocities. Wall shear rates were calculated as previously described (Weckbach et al., 2014 (link)).

Blood samples were collected from the anterior cephalic vein after the dogs were subjected to fasting for at least 16 h at the beginning (0 weeks) and the end (16 weeks) of the experiment. The collected blood was immediately divided between EDTA vacutainer tubes (ref 367861, BD Vacutainer, NJ, USA) and serum vacutainer tubes (ref 367812, BD Vacutainer). The blood collected in the EDTA tubes were used in the complete blood cell count (CBC) analysis using an automated blood analyzer ProCyte Dx (IDEXX Laboratories, Westbrook, ME, USA) immediately after collection. The blood collected in the serum vacutainer tubes were centrifuged at 1,650×g for 15 min to separate the serum; the serum supernatant was stored at –80°C until further use. Serum biochemical composition analysis was performed using an automated biochemistry analyzer, Hitachi 7180 (Hitachi High-Technologies, Tokyo, Japan). Adiponectin (MBS2607889, MyBioSource, San Diego, CA, USA), immunoglobulin G (IgG, ab157701, Abcam, Cambridge, MA, USA), interleukin-6 (IL-6, ab193686, Abcam), insulin (MBS031096, MyBioSource), leptin (MBS037935, MyBioSource), and tumor necrosis factor-α (TNF-α, ab193687, Abcam) were measured by enzyme-linked immunosorbent assay using a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) in duplicates, according to the manufacturer’s instructions.