A0136

Immunohistochemical techniques were performed, according to standard protocols. Briefly, frozen tissue sections were fixed, cryostat sectioned and stained with a rabbit anti-human C1q antibody (A0136; Dako), a goat anti-human C3 antibody (sc-20137; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), a rabbit anti-human CR2 (HPA052942, Sigma-Aldrich, St. Lous, MO, USA) or with respective isotype control antibodies, and then washed and incubated with HRP-conjugated anti-rabbit or anti-goat IgG secondary Abs. Afterwards, tissue slides were incubated with DAB substrate (Dako) and counterstained with Mayer`s hemalum solution.

Rabbit anti-human SAP polyclonal antibody (pAb) (565191, Calbiochem), rabbit anti-human CRP pAb (235752, Calbiochem), rabbit anti-human C1q pAb (A0136, Dako), mouse anti-human complement component C5b-9 monoclonal antibody (mAb) (IgG2a) (011-01, Antibody Shop), mouse IgG1 isotype control (BD Biosciences), mouse IgG2a isotype control (BD Biosciences), rabbit IgG isotype control (Invitrogen), FITC-conjugated goat anti-rabbit pAb (F1262, Sigma-Aldrich), FITC-conjugated goat anti-mouse pAb (F0479, Dako), mouse anti-human CD45 FITC/CD14 PE (342408, BD Biosciences), mouse anti-human CD11b APC-Cy7 (560914, BD Biosciences), Alexa-555-conjugated goat anti-mouse IgG (A-21424, Thermo Fischer), biotinylated goat anti-rabbit IgG (BA-1000, Vector Laboratories), or biotinylated goat anti-mouse IgG (BA-9200, Vector Laboratories) are the commercial antibodies.

Immunohistochemistry was used to investigate intracerebral IgG and IgM distribution. Briefly, paraffin wax-embedded tissues from humans or macaques were dewaxed in xylene and hydrated through graded alcohols. Endogenous peroxidase activity was suppressed by 3% H₂O₂ in PBS. Subsequently, sections were incubated with goat anti-IgG (anti-human IgG (gamma chain), 1/100, SAB3701291, Sigma-Aldrich, Saint-Louis, MI, USA), with rabbit anti-IgM (anti-human IgM, mu chain, 1/250, A0425), with polyclonal rabbit anti-Human C1q (1/100, A0136, Dako, Les Ulis, France), with anti-MBP (1/100, SMI94R BioLegend, San Diego, CA, USA), with rabbit anti-IBA1 (1/100, ab178846, Abcam, Cambridge, UK), or with mouse anti-CD68 (1/150, ab 955, KP1, Abcam). Tissues were then incubated with secondary antibodies coupled to fluorochromes Alexa 488 or Alexa 594 (ThermoFisher, Villebon-sur-Yvette, France). Alternatively, biotinylated rabbit-anti-goat, rabbit-anti-mouse, or goat-anti-rabbit antibodies were used as secondary antibodies, for 30 min at room temperature, followed by the avidin-biotin-peroxidase complex (Vectastain Elite ABC Kit, Vector Laboratories, PK 6100; Burlingame, CA, USA). Positive antigen-antibody reactions were visualized by incubation with 3,3-diaminobenzidine-tetrahydrochloride (DAB)–H₂O₂ in 0.1 M imidazole, pH 7.1 for 5 min, followed by slight counterstaining with hematoxylin.