Dmc2900 cmos camera

Mice were treated by intrascrotal injection of 500 ng TNF-α (R&D Systems) 2 hours prior to microscopy, anesthetized, and prepared for intravital microscopy, as described (60 (link)). Movies from cremasteric postcapillary venules ranging from 20 to 40 μm in diameter were recorded using BX51WI microscope (Olympus) with a water immersion objective ×40, 0.80 NA, and an Olympus charge-coupled device camera (CF8/1, Kappa). Blood samples were taken after the experiments, and WBC and neutrophil counts were determined using ProCyte Dx Hematology Analyzer (IDEXX). Rolling velocity and leukocyte adhesion efficiency (number of adherent cells/mm² divided by the systemic neutrophil count) were calculated on the basis of the recorded movies using Fiji software (61 (link)). Afterward, cremaster muscles were fixed with 4% PFA (AppliChem) and stained using Giemsa (MilliporeSigma). The number of perivascular cells/mm² was calculated with a Leica DM2500 microscope equipped with a DMC2900 CMOS camera and an HCX PL APO 100×/1.40 Oil Ph3.

Retinas were digested using trypsin as previously described with slight modifications [27 ]. Briefly, after enucleation the eyecup was fixed in 4% PFA for 45 min, then the retina was isolated and further fixed in 4% PFA overnight. The next day, the retinas were washed (5x) with distilled water. After overnight shaking in water at room temperature, retinas were transferred into 24-well plates and incubated in 3% trypsin (#9002-07-7, Affymetrix USB, Ohio, USA) in 0.1 M Tris buffer (pH 7.8) at 37 °C for 90 min. The inner limiting membrane was removed with scissors and then the vasculature isolated by several washing steps. The retinal vasculature system was flat mounted on Thermo Scientific™ SuperFrost Plus™ slides and stained with H&E (H&E fast staining kit, #9194.1, Carl Roth). Microscopy was performed at the Core Facility Bioimaging of the Biomedical Center at the LMU with a Leica DM6 FS microscope. Bright field images were recorded with Leica DMC2900 CMOS camera with an image pixel size of 145 nm. A 20x/0.8 objective was used for quantification and overview image of the network and a 40x/0.95 objective for detailed view of the vascular network.