Eclipse ti 5

LNs were collected and embedded in OCT Compound (Tissue-Tek). Cryostat sections (6 um thick) were mounted on Poly-lysine microscope slides (Thermo Scientific). Brightfield and DiD images of cryostat sections were acquired using Nikon Eclipse Ti-5 before acetone fixation. Sections were subsequently dried and fixed in cold acetone for 5 min at −20° C, blocked with 3% BSA in PBS blocking buffer at room temperature for 30 min, and incubated with anti-CD169 (ThermoScientific) and SIGNR1 (eBioSciences) antibodies diluted in the blocking buffer for 1 hour. Primary antibodies were detected with Goat-anti-Rat-AF488 (against CD169, BioLegend) and Goat-Anti-Hamster IgG-568 (against SIGNR1, Abcam). Images were acquired with the Nikon Eclipse Ti-5 microscope, using 4× objective lens and NIS-Elements D3.2 Software and processed with Fiji software.

Coronal sections (2 mm anterior to bregma) were selected and processed for immunohistochemistry staining. Staining was done as previously described (12 (link)). Briefly, sections were removed from −80°C and thawed. The fresh frozen sections were fixed with 4% PFA for 15 min. After being further washed three times in PBS containing 0.1% Triton X-100, they were incubated with 10% Block ACE (AbD Serotec) in PBS for 1 h at room temperature. Then sections were incubated in PBS/0.3% BSA solution containing primary antibodies anti-Ki67 (proliferative cell marker, 1:100, Abcam) and anti-PDGFRα (OPC marker, 1:100, Santa Cruz) at 4°C overnight. After washing with PBS three times, they were incubated with secondary antibodies (1:200; Jackson Immunoresearch Laboratories) for 1 h at room temperature. Similarly, sections were stained with anti-Ki67 and anti-nestin antibodies (NSPC marker, 1:100, Abcam). Then the sections were washed three times and covered with VECTASHIELD mounting medium with DAPI (Vector Laboratories). Immunostaining was analyzed with a fluorescence microscope (Nikon Eclipse Ti-5).