Lsm780 meta confocal laser microscope

Colon tissues were dissected from sacrificed mice, flushed and cleaned with PBS, and cut open longitudinally. The tissues were then fixed in 4% paraformaldehyde (ACROS organic, Waltham, MA) for 24 hr, washed with PBS, incubated in 20% sucrose in PBS for another 24 hr. Tissues were then flash-frozen in optimal cutting temperature compound (OCT-Sakura, Torrance, CA) in dried ice-chilled 100% ethanol. The frozen tissue blocks were stored in –80°C until use. Six mm-thick frozen sections were cut with a cryostat and allowed to dry overnight at room temperature. Sections were then washed with PBS, rinse briefly with water, and then mounted with VECTASHIELD mounting medium containing anti-fading agents with DAPI (Vector laboratories, Burlingame, CA). The tissues were examined under a LSM780 Meta confocal laser microscope (Carl Zeiss, Germany). The captured images were viewed and analyzed using Zeiss Zen Meta imaging 2012 software. Scale bar was calculated with ZEN software then extrapolated to all images with the same magnification power.

Total bone marrow cells from naïve C57BL/6 mice were enriched and CD11b⁺, CD68⁺, F4/80⁺ macrophages were consecutively sorted three times by magnetic bead isolation (Miltenyi Biotech) to achieve >95% purity. Purified macrophages were labeled with CFSE (Molecular Probes, Eugene, OR), a green fluorescent cell staining dye, as described previously (Sharma et al., 2010 (link)). A total of 6×10⁵ cells/mouse were injected via the tail vein immediately after sham/TBI injury. To provide spatial analysis of macrophage trafficking into the brain, deeply anesthetized mice were perfused with PBS, followed by fixation with 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Brains were post-fixed overnight in paraformaldehyde followed by cryoprotection with 30% sucrose (pH 7.4) until brains permeated. Serial coronal sections (12 µM) were prepared using a cryostat microtome and directly mounted onto glass slides. Anti-fade mounting media was added and glass cover slips were placed atop the slide. CFSE immunofluorescence was imaged using a LSM780 Meta confocal laser microscope and vendor supplied software (Carl Zeiss).