Coolsnap ez camera

Uninflamed control skin or chronically inflamed mouse skin was harvested 3 weeks after induction of inflammation with CFA, fixed for 6 hours in 4% PFA in PBS and incubated overnight in 30% sucrose before freezing in OCT. To reduce non-specific staining, 6-8 μm thick skin sections were blocked with 10% donkey and 40% goat serum. B cells were visualized with polyclonal goat anti-mouse CD20 (Santa Cruz Biotechnology), which was labeled with CF594 or CF488 Mix-n-Stain antibody labeling kits according to the manufacturer's instructions (Biotium). Tissue sections were additionally stained with rat anti-mouse CD31 (MEC13.3; BD Biosciences) or IL-10 (JES5-16E3; BD Biosciences). Multi-species adsorbed F(ab)₂ donkey anti-rat IgG conjugated with Alexa Fluor 488 or 594 (Jackson ImmunoResearch) were used as secondary antibodies and DAPI (Invitrogen) to visualize nuclei. Specificity of the CD20 and IL-10 staining was confirmed by including tissues from Rag1^–/– and Il10^–/– mice, respectively (data not shown). Sections were mounted with Prolong Gold Antifade (Invitrogen) and images acquired on a Nikon Eclipse E600 microscope using a Photometrics CoolSNAP EZ camera and NIS-Elements BR 3.0 software.

Following intracardiac perfusion with Tyrode’s and 4% paraformaldehyde (PFA), spinal columns were removed, post-fixed for 3 days in PFA, decalcified in 0.5 M EDTA for 4 days, cryoprotected in 30% sucrose, and embedded and frozen in OCT (Cellpath). 12 µm sections were blocked with Avidin B and biotin (Vector Labs) before staining with the following primary and secondary antibodies: goat anti-MBP (Santa Cruz Biotechnology), Alexa Fluor 488-conjugated donkey anti-goat IgG (Life Technologies), rat anti-CD45 (eBiosciences), Alexa Fluor 647-conjugated goat anti-rat (Life Technologies), mouse anti-SMI-32 (Covance), and Alexa Fluor 594 conjugated goat anti-mouse IgG. DAPI (Life Technologies) was used to label nuclei. Images were acquired on a Nikon Eclipse Ti, CoolSnap EZ camera, and NIS Elements: Basic Research v3.10. Appropriate image processing, including image merging and black level and brightness adjustments, was performed in Photoshop CC 2017 and applied equally to all samples.