Intravital Microscopy of TNF-α-Induced Inflammation

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.