Cd31 alexa fluor 647

For serial intravital microscopy of the calvarium, bone marrow B cell precursors (Lineage⁻ CD93⁺) from dsRed reporter mice and mature B cells from the spleen (CD19⁺ IgD⁺) were FACS-sorted using a FACSAria II cell sorter (BD). For B cell progenitor imaging experiments, 10⁶ cells were injected i.v. into non-irradiated recipient C57BL/6 mice one day prior to stroke induction. To visualize mature B cell accumulation in the bone marrow, 5×10⁶ cells were injected i.v. one week prior to surgeries. To highlight bone architecture, OsteoSense® 750EX, a fluorescent bisphosphonate imaging agent (Perkin Elmer), was administered i.v. 24 hours prior to imaging (4 nmol/mouse, PerkinElmer). To outline the vasculature, 15μg CD31-Alexa Fluor 647 (102516, BioLegend) 30 min prior to imaging. In vivo imaging was performed using a confocal microscope (IV100 Olympus). Z-stack images for each location were acquired at 2μm steps, and post-processing was performed using Image J software (NIH).

For immunofluorescence staining, cells in the device were fixed using
4% PFA for 15 minutes at room temperature (RT), permeabilized with
Triton X-100 (0.15% v/v in PBS) for 15 minutes, and then blocked in
bovine serum albumin (BSA 3% w/v in PBS) for 1 h at room temperature.
Monoclonal antibodies specific for human ZO-1 (Alexa Fluor 488) were purchased
from Thermo Fisher, CD31 (Alexa Fluor 647) were purchased from BioLegend and
Hoechst 3342 were purchased from Molecular Probes. Mouse monoclonal antibodies
specific for ICAM-1 (Alexa Fluor 488) were purchased from BioLegend. All the
samples were washed three times and stored in PBS before imaging.