Immunohistochemistry

Snap-frozen blocks of post-mortem normal control and MS brains were cut (6 μm) on a cryostat and mounted on superfrost plus glass slides (VWR international, Belgium). Sections were stored at −80°C. To determine MS lesion type, one section per case was stained for proteolipid protein (PLP, 1:500, Serotec, Kidlington, UK) and one for human leukocyte antigen–antigen D related (HLA-DR; MHCII, 1:2,000, in house antibody). Sections were defrosted at room temperature (RT), where after they were fixed in acetone for 10 min. After 3 washes with PBS, slides were incubated with antibodies for 1 h at RT. All antibodies were diluted in 1x phosphate-buffered saline (PBS) and 0.05% Tween-20. After 3 washes with PBS, slides were incubated with EnVision+ Dual Link System-HRP (Agilent DAKO, Santa Clara, CA, USA) for 30 min. 3,3′-diaminobenzidine (DAB) was used as a chromogen. Sections were counterstained with haematoxylin for 1 min and thoroughly washed with tap water for 3 min. Finally, sections were dehydrated in alcohol and xylene series and mounted with Entellan. Bright field images were taken with a Zeiss microscope (AXIO Scope A1, Carl Zeiss, Germany).

To reveal the cellular localization of MC4R, immunofluorescence triple-labeling was performed. Slides were fixed in acetone for 10 min and a blocking step was performed (30 min 10% normal goat serum). Thereafter, slides were incubated with the antibodies for MC4R (1:1,000, ab75506, Abcam, Cambridge, UK) and HLA-DR (MHCII) or biotinylated ULEX Europaeus Agglutinin1 (UEA-1) (1:1,000, Vector labs, Burlingame, CA, USA). Slides were washed and incubated with sheep-anti-rabbit alexa-488, sheep-anti-mouse-alex647 (1:400, Invitrogen, Carlsbad, CA USA), Cy3 labeled GFAP (1:700, Sigma-Aldrich), or streptavidin-647 (1:400, Invitrogen), respectively, diluted in 1x PBS/0.05% Tween-20 at RT for 1 h. After three washes with PBS, slides were incubated for 1 min with Hoechst (1:1,000) to visualize cellular nuclei and mounted with Mowiol. Z-stack images were taken with a 40x objective with a confocal microscope (Leica SP8 STED, Leica microsystems, Germany).