M3 84

Spinal cords and spleens of EAE mice were removed following perfusion with 4% (wt/vol) PFA and post-fixed for 2–3 h. After embedding in paraffin, 4 μm thin sections were prepared by using a microtome. For immunohistochemistry, αCD3 (1:200, MCA1477, Bio-Rad) and αMac-3 (1:200, M3/84, BD Pharmingen) antibodies were used to detect immune cells. Luxol Fast Blue staining was performed for evaluation of demyelination and Bielschowksy silver impregnation for axonal integrity/damage. Quantification of axonal preservation, cellular infiltrates, and degree of demyelination was performed in a blinded fashion on 9 independent spinal cord sections per mouse. Cellular infiltrates were quantified per square millimeter of white matter by overlaying a stereological grid onto sections and demyelinated areas were determined semi-automatically by CellP Software (Olympus). Six visual fields of the cervical, thoracic, and lumbar spinal cord were used for quantification of axonal preservation counted on a 100 μm diameter grid.

Mouse EAT was obtained and fixed in 4% (wt/vol) paraformaldehyde in PBS for 3h and subsequently transferred to 30% (wt/vol) sucrose overnight. The samples were then embedded in paraffin and serially sectioned (6 μm). Cross-sections were prepared for immunohistochemical staining overnight at 4 °C with a 1:100 dilution of a mouse mAb to Mac3 (M3/84; BD Biosciences), 1:400 dilution of a rat anti-mouse CD11c (GB11059, Servicebio), and 1:2000 dilution of a rat anti-mouse CD206 (GB13438, Servicebio). Immune complexes were detected with biotinylated secondary antibodies (BD Biosciences), HRP-conjugated streptavidin (Dako), and the peroxidase substrate diaminobenzidine (Dako). Images were captured using a microscope (Leica, Germany). Macrophage in EAT was quantitated by calculating the ratio of nuclei of Mac3-positive cells to total nuclei in 10 fields of 3 slides for each individual mice using 6 mice for each group [22 (link)]. The CD11c and CD206 staining level were validated quantitatively by image analysis assessing the pixel values on the digital slide images. In some experiments, cross-sections were stained with hematoxylin-eosin (HE). For quantification of adipocyte area, 5–6 fields per section were averaged and 6 mice per group were calculated. Image J software (NIH, USA) was used to measure adipocyte area and shown as the average adipocyte area (in μm²).

For detection of VV-infected cells or macrophages, paraformaldehyde (PFA) (4%) fixed and paraffin embedded CNS tissue sections were incubated with Bond Primary Antibody Diluent (Leica) containing either polyclonal rabbit anti-VV serum (1:1000; Quartett Immunodiagnostika & Vertriebs-GmbH, Berlin) or monoclonal antibodies against Mac-3 (1:750; M3/84) purchased from BD Pharmingen. IHC staining was performed on an automated Leica BOND-MAX instrument using Bond Polymer Refine Detection Solution for DAB. For detection of GFAP, PFA-fixed and paraffin-embedded CNS sections were incubated with Dako polyclonal rabbit anti-GFAP antibodies (Z0034; 1:13000) in Ventana buffer and staining was performed on a Ventana NexES IHC Slide Stainer using iVIEW DAB Detection Kit (Ventana). Images were taken using the Leica SCN400 slide scanner analysis software or were acquired on an Olympus BX53 Microscope (DP72 camera) using the cellSens 1.8 digital imaging software (Olympus).