Nis elements ar analysis 5

Confocal microscopy imaging was performed as described previously (24 (link)). Mice were anesthetized (s.c.) with ketamine and xylazine (Syntec, 60 mg/kg and 15 mg/kg, respectively). Before surgery mice received a single dose of IgG (12,5 µg/g, Sigma-Aldrich) and after fifteen minutes they received a mixture of the following antibodies or fluorescent probes: anti-aCD31 BV421 (0,15 µg/g, clone 390, BD), anti-F4/80 BV480 (0,05 µg/g, clone T45-2342, BD), anti-Ly6C BV605 (0,1 µg/g, clone AL-21, BD), anti-aLy6G BV711(0,1 µg/g, clone 1A8, BD), anti-CD19 BB515 (0,1 µg/g, clone 1D3, BD), anti-NK1.1 PE (0,06 µg/g, clone PK136, BD) and anti-CD3e APC (0,06 µg/g, clone 145-2C11, BD) - all from BD Biosciences. In neonatal mice, delivery of drugs (via eye venous plexus) and surgery were made with minor modifications. All images were acquired using an inverted Nikon Eclipse Ti coupled to an A1R confocal microscope loaded with a spectral detector and XYZ motorized stage. Different fields of the organ were pictured. Blood flow analyses was done after FITC-albumin injection (12,5 µg/g, Sigma-Aldrich; i.v.) using resonant scanner (no time lapse). Images and videos rendering were made using NIS-Elements AR Analysis 5.41.01(Nikon) and Volocity(6.3) (Perkin Elmer, Waltham, MA).

For each animal, three images were selected and submitted to cell counting. Each image was cropped and enlarged, allowing a reliable cell analysis and quantification of each field. This analysis was done manually using ImageJ. The average amount of cell types from the three images, for each animal of each group was plotted on the graphs. Cellular tracking, morphologic analysis and area measurement were performed semi automatically utilizing NIS Elements AR Analysis 5.41.01 (Nikon). Movies were recorded taking one frame every 30s. Tracking analysis was performed using only one animal.