Application suite las advanced fluorescence lite software

All data are presented as the means ± standard deviations of the means (SD). The 3D image overlays were visualized using Leica Application Suite (LAS) Advanced Fluorescence Lite software (LAS AF Lite, 2.4.1 build 6384, Leica). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the immunohistochemistry and immunofluorescent results. An experimenter who was blinded to the experimental condition analyzed all sections. A two-way repeated measures ANOVA with multiple comparisons was performed to compare the BBB permeability measurements. An independent-samples t test was used for dual comparisons. Means were compared using one-way ANOVA analysis followed by a post hoc Tukey's multiple comparison test for multiple comparisons. Statistical analyses were completed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). A P value < 0.05 was considered statistically significant.

Recipients were transfused via lateral vein with PBS or 50 μl of packed KEL-DiO RBCs diluted to 300 μl total volume in PBS. 1, 3, 5, and 7-days post transfusion, spleens were quick frozen in isopentane on dry ice using TissueTek optical cutting temperature freezing medium (VWR Scientific, Randor, PA). 7 μm thick frozen sections were cut and fixed in acetone for 20 min at −20°C. Fixed sections were then washed 3 times in PBS and incubated in 0.5% blocking buffer [PBS + 0.5% fetal bovine serum (FBS)] for 2 h at room temperature. The blocking buffer was then removed and sections were stained with PE anti-mouse CD1d + Alexa Fluor 647 anti-mouse IgD diluted in blocking buffer for 1 h at room temperature. Sections were then washed 3 times with PBS and mounted using Prolong Gold anti-fade mountant (ThermoFisher Scientific, Waltham, MA). Images were acquired using the HC Plan Fluotar 10X (0.3 NA air, WD 11.0 mm) or HC Plan APO CS2 40X (1.3 NA oil, WD 0.24 mm) objective on a Leica SP8 multiphoton confocal microscope. Images were then analyzed using the Leica application suite (LAS) Advanced Fluorescence lite software.