Anti mda

Animals were anesthetized with pentobarbital and perfused with ice-cold PBS followed by cold 4% paraformaldehyde. Spinal cords were placed in OCT and deep frozen in dry ice. Serial 10 μm longitudinal sections of spinal cords were prepared, and immunofluorescence stainings were performed as previously described (59 (link)). The following antibodies were used: anti-CD3 (Bio-Rad Laboratories, MCA1477), anti-IBA1 (Fujifilm Wako, 019-19741), anti-iNOS (Novus Biologicals, NBP2-22119), anti-NEUN (MilliporeSigma, MAB377), anti-APC (Ab-7, Calbiochem, OP80), anti-MDA (Abcam, ab6463), anti-GFAP (Dako, Z0334), anti-NG2 (MilliporeSigma, AB5320), anti-MPB (MilliporeSigma, MAB386), and anti-NF M (Chemicon International, AB1987). Goat anti–rabbit Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, A11034), goat anti–mouse Alexa Fluor 488 (Invitrogen, Thermo Fisher Scientific, A11001), goat anti–rabbit Alexa Fluor 555 (Invitrogen, Thermo Fisher Scientific, A27039), goat anti–rat Alexa Fluor 555 (Invitrogen, Thermo Fisher Scientific, A21434), and goat anti–mouse Alexa Fluor 555 (1:200, Invitrogen, Thermo Fisher Scientific, A28180) were used as secondary antibodies appropriately. Nuclear counterstain was performed using DAPI (Vector Laboratories). Bielschowsky silver impregnation (for axons) and Gold-Black (for myelin) staining were performed as previously described (60 (link)).

C‐MSC were plated on 1.8 cm² chamber slides (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a density of 20,000 cells/cm². After 24 h of culture in basal conditions, C‐MSC were washed with PBS and fixed in 4% paraformaldehyde in PBS. After the blocking step in 10% goat serum (Sigma‐Aldrich, St. Louis, Missouri, USA), cells were incubated with primary antibodies anti‐MDA (1:2,500; Abcam, Cambridge, UK) and anti‐CD36 (1:200; BD, Franklin Lakes, New Jersey, USA) at 4°C overnight. After washing, sections were incubated with the fluorochrome‐conjugated antibody goat anti‐rabbit IgG Alexa 488 1:200 (Alexa Fluor, Waltham, Massachusetts, USA) for 1 h at RT in the dark. Nuclear staining was performed by incubating sections with Hoechst 33342 (1:1,000; Life Technologies, Carlsbad, California, USA). Sections were observed by Zeiss Axio Observer.Z1, with Apotome technology, and images were acquired with the software AxioVision Rel. 4.8. For each dish, 15 fields were examined.

Human ventricular samples were fixed in 4% paraformaldehyde (Santa‐Cruz, Dallas, Texas, USA) in phosphate‐buffered saline (PBS; Lonza, Basel, Switzerland) and processed for paraffin embedding. Paraffin‐embedded sections (6 μm thick) were de‐waxed in xylene and rehydrated in ascending alcohols. The immunofluorescence analysis was performed following antigen retrieval with incubation with target retrieval solution citrate pH6/microwave (Dako, Santa Clara, California, USA). Sections were incubated with primary antibody anti‐MDA (1:2,500; Abcam, Cambridge, UK) and anti‐CD36 (1:200; BD, Franklin Lakes, New Jersey, USA) at 4°C overnight (see Appendix Table S7). After washing, sections were incubated with the fluorochrome‐conjugated antibody goat anti‐rabbit IgG Alexa 488 1:200 (Alexa Fluor, Waltham, Massachusetts, USA) for 1 h at room temperature (RT) in the dark. Nuclear staining was performed by incubating sections with Hoechst 33342 (1:1,000; Life Technologies, Carlsbad, California, USA). Sections were observed by Zeiss Axio Observer.Z1, with Apotome technology, and images were acquired with the software AxioVision Rel. 4.8. For each explanted heart subject, three consecutive slices and at least five fields for each slice were examined, excluding autofluorescence and aspecific signals. For ACM biopsy samples, all the samples were sliced and examined.