Fluoropure grade

Four-µm-thick tissue sections prepared from formalin-fixed and paraffin-embedded tissue samples were deparaffinized in xylene twice for 10 min each and rehydrated in a graded ethanol series for 10 min each. After blocking endogenous peroxidase by 3% hydrogen peroxide and antigen retrieval by 14 min of microwave heating in EDTA buffer (pH 7.4), the samples were incubated with primary antibodies against CD8 (1:400, ab199016, Abcam, Cambridge, UK), CD103 (1:200, ab129202, Abcam), and CD69 (1:200, bs-2499R, Bioss, Woburn, MA, USA) overnight at 4 °C, and then incubated with peroxidase-linked secondary antibody for 30 min at room temperature. The samples were immersed in 3, 3-diaminobenzidine (DAB) and counterstained with Mayer’s hematoxylin, followed by dehydration in a graded ethanol series for 10 min each. After mounting, the samples were subjected to microscopic observation. For fluorescence immunohistochemical staining for CD8 and CD103, Alexa 488 Anti-mouse (1:500, A11017, Invitrogen, Tokyo, Japan) or Alexa 647 Anti-Rabbit (1:500, A21245, Invitrogen) was used as the secondary antibody, and DAPI (D21490, FluoroPure™ grade, Invitrogen) was used for DNA staining.

BOND-MAX staining methods have been previously described (21). The following antibody working solutions were prepared and loaded into the BOND-MAX Processing Module: a) 10 μg/mL rabbit monoclonal anti−cleaved caspase-3 antibody (R&D systems, MAB835, clone 269518, Milan, Italy) prepared in Bond Primary Antibody Diluent (Leica Biosystems, Buffalo Grove, IL), and goat-anti-rabbit Alexa Fluor 546 antibody (Invitrogen, Life Technologies, Grand Island, NY) prepared 1:100 in 1× Bond Wash Solution; b) 5 μg/mL monoclonal anti−phospho (Ser 139) γH2AX-FITC conjugate (Millipore, 16-202a, clone JBW301, Billerica, MA) antibody prepared in Bond Primary Antibody Diluent (Leica Biosystems, Buffalo Grove, IL); and c) 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride, FluoroPure™ grade (Invitrogen, Life Technologies, Grand Island, NY) prepared at 0.25 μg/mL in Bond Primary Antibody Diluent.

The exosomes isolated from NPCs were labeled by PKH26 membrane dye (Sigma-Aldrich MINI26, USA) before being applied to the culture medium for NPC cells or HUVECs in order to observe the cellular uptake of labeled exosomes. The labeling methods were performed according to the instructions of the manufacturers. After 3 hours coculture with labeled exosomes (20 μg total protein for each 35 mm petri dish according to BCA assay), treated cells were fixed with 4% paraformaldehyde (PFA) and labeled with phalloidin-FTIC (Sigma-Aldrich P5282, USA). Then, 4',6-diamidino-2-phenylindole (DAPI, FluoroPure™ grade, Invitrogen) was applied to stain the nuclei and the samples were imaged by a Leica SP8 confocal microscope to confirm the internalization and intracellular distribution of labeled exosomes. The 3D images were remodeled from z-stack series with the 3D viewer module of Las X software.

The culture medium of H9c2 cells transfected with the LBH-eGFP plasmid or pEGFP-C1 plasmid was collected for exosome isolation. The isolated exosome samples were subjected to nanoflow cytometry to detect potential LBH-eGFP fusion protein or supplemented into the culture medium for CFs to observe cellular uptake of the labeled exosomes. For simulation of the entire crosstalk process without exosome isolation, the coculture of LBH-eGFP-transfected H9c2 cells and rat CFs was implemented by Transwell inserts (Corning #3450, 0.4 μm pore size) and glass bottom 6-well plates (Nest #801004). Also, the exosomes isolated from mouse CMs were labeled with PKH26 membrane dye (Sigma-Aldrich MINI26, USA) before being applied to the culture medium for CFs. After 6 hours of coculture with exosomes (20 μg total protein for each 35 mm petri dish according to BCA assay) or 24 hours of coculture with H9c2 cells, the treated CFs were fixed with 4% paraformaldehyde (PFA), stained with phalloidin-rhodamine (Santa-Cruz PHDR1, USA)/phalloidin-FTIC (Sigma-Aldrich P5282, USA) and 4′,6-diamidino-2-phenylindole (DAPI, FluoroPure™ grade, Invitrogen), respectively, and imaged by a Leica SP8 confocal microscope to confirm the internalization of labeled exosomes and the intracellular distribution of LBH-eGFP. The 3D images were remodeled from z-stack series with the 3D viewer module of Las X software.