Entrance of B cells into LN follicles. (A) Recently arrived B cells avoid the LN follicle. Fluorescent nanodots were used to outline HEVs. Adoptively transferred B cells (green) were tracked between 40 minutes and 2 hours after transfer by TP-LSM in the vicinity of a LN follicle defined by labeled B cells (red) transferred 24 hours earlier. Individual tracks are shown in different colors. Images acquired every 15 seconds, 21 slices separated by 6 μm. (B) Many Gnai2^−/− B cells fail to access the LN follicle. Wild-type (green) and Gnai2^−/− (red) B cells were adoptively transferred 2 days before imaging. Red channel and green channel from the same image are shown. The lymphatics were outline by injecting labeled antibody against LYVE-1 (white) subcutaneously near the tail base. (C) B cells enter the LN follicle from the base. Three separate mice imaged at 150 minutes, 300, and 440 minutes after transfer of B cells (green). Follicles outlined with white circle based on the location of B cells (red) transferred the day before. Shown are images from 3 slices for each time period located at the base, at 40 microns, and 80 microns above the base. (D) Location of newly entering B cells in the LN follicle. The ratio of newly transferred to previously transferred B cells in base region, 40 microns, and 80 microns closer to the capsule is shown. The 3 ratios were summed. The results are from the analysis of imaging 8 follicles. (E) Tracking B cells entering the LN follicle. Newly-transferred B cells (green) were imaged by intravital TP-LSM for 1 hour starting at 3 hours 30 minutes after transfer every 15 seconds. For each time point, 21 slices were collected at 6 μm intervals. B cells (red) were transferred 24 hours before imaging to outline the follicle. Individual cell tracks are shown of B cells entering the LN follicle (outer circle) and crossing into the center region (dotted circle). Scale bars are 50 μm.