Citrate ph 6 microwave

Human ventricular samples were fixed in 4% paraformaldehyde (Santa Cruz) in PBS (Lonza) 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 pH 6/microwave (Dako). Sections were incubated at 4°C overnight with primary antibodies for the detection of mesenchymal surface markers (see Supplementary Table S2), namely, anti-CD29 (1:40; Leica), anti-CD44 (1:200; Abcam), and anti-CD105 (1:100; Abcam) diluted in 2% goat serum (Sigma–Aldrich). After washing with PBS, sections were incubated for 1 h at RT in the dark with proper secondary antibodies (see Supplementary Table S3). Nuclear staining was performed by incubating sections with Hoechst 33342 (1:1000; Life Technologies). Sections were observed by Zeiss Axio Observer.Z1, with Apotome technology, and images acquired with the software AxioVision Rel. 4.8. For each explanted heart patient, five slices and at least 10 fields for each slice 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.