Dapi stain

Primary mixed glial cultures were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 10 min and then washed with PBS. Cells were then blocked for 1 h at room temperature (RT) in blocking solution consisting of 5% normal donkey serum in PBS containing 0.1% Triton X-100 (PBST). Primary antibodies rabbit anti-GFAP 1:10,000 (abcam ab7260) and rabbit anti-Iba-1 1:100 (Wako LKR1186) were then added in blocking solution overnight at 4 °C. The following day, cells were washed with PBS and then incubated in secondary antibodies (anti-rabbit or anti-mouse Alexa Fluor® 488 or Alexa Fluor® 596 raised in donkey, by Thermo Fisher, used at 1:1000) in blocking solution for 2 h at RT. After another set of PBS washes, a DAPI stain was applied (Sigma-Aldrich, 1:2000) to stain nuclei and then cells were mounted with Mowiol mounting media and stored at 4 °C. Imaging was performed using a Leica inverted epifluorescence light microscope and Leica Application Suite software. The proportion of cells that were GFAP^+ve or Iba1^+ve were determined using MetaMorph Image Analysis Software (Molecular Devices, CA, USA) by using the number of DAPI-labelled nuclei as the total number of cells in the culture.

Acceptor cells were seeded 24 h before the uptake experiment, at 200,000 cells per well in a 24-well plate on the top of coverslips. Acceptor cells were incubated with GFP-Hsp70 EVs (10 μg protein/mL) for 24 h at 37 °C). Then cells were washed with PBS, fixed 15 min RT with PBS 4% Paraformaldehyde. After washing with PBS, samples were treated with 0.2% Triton X100 and incubated with DAPI stain (Sigma Aldrich, Missouri, USA) and WGA-Alexa⁶³³ (Thermo Scientific, Illinois, USA) for 15 min to respectively label nucleus and plasma membrane (Supplementary Fig. 1), later delineated with cyan and magenta lines (Fig. 1D). Samples were then mounted and imaged using a SP8 confocal microscope (Leica Microsystems, Germany). WT acceptor HeLa cells not treated with fluorescent EVs, and virtually negative for GFP fluorescence, were used as negative control to set up the threshold to specifically detect GFP-foci emanating from donor EVs, when acceptor cells of interest were treated by GFP-HSP70 EVs. Fluorescent foci within cells were manually counted.

Immunolocalization was performed in the following groups: Group (I) WCtrl (wound control), Group (II) VCtrl (vehicle control), Group (III) infected wound (received with 0.5 mg/kg) PaTx-II, Group (IV) (positive control), and 2% fusidic acid ointment (0.5 mg/kg) treated wound as a standard. Mice were sacrificed with excess exposure of carbon dioxide (CO₂) inhalation and wound tissues fixed in 30% Bouins fluid. Paraffin sections (7 μm) of groups were incubated overnight at 4 °C with antibodies for type I collagen (PanCytokeratin), tumor necrosis factor-alpha (TNF-α), or cyclooxygenase-2 (COX-2) at a dilution of 1:500 in PBS with 1% bovine serum albumin (BSA) (Santa Cruz Biotechnology Inc, Santa Cruz, California, USA), followed by 1 h incubation at 28 °C. The secondary antibody (polyclonal anti-rabbit IgG HRP) was incubated at 1:5000 dilution in PBS with 12% BSA. Subsequently, sections were washed in PBS containing 0.1% Tween-20, before mounting with 0.5 µg/mL of DAPI stain (Sigma, St Louis, Missouri, USA) and photographed on an Olympus fluorescence microscope [85 (link)]. Sectioning with 3,3-diaminobenzidine (DAB) staining was also performed for collagen (type I).

For all experiments involving RD cells, the cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for seven minutes at room temperature prior to permeabilization with 0.5% Triton X-100 (Sigma) in PBS. Cells were then incubated with 1% BSA in PBS block solution for 30 minutes at room temperature and then incubated overnight with J2 or K1 antibody at a concentration of 2 μg/ml at 4°C. Appropriate FITC- or TRITC-conjugated secondary antibodies (Jackson ImmunoResearch) and DAPI stain (Sigma) were then used prior to visualization. For the human myoblast experiment, conditions were essentially the same, except that cells were fixed with 2% paraformaldehyde and co-incubated overnight with J2 or K1 and anti-DUX4 (E5-5) antibody. Cells were imaged on a Zeiss AxioPhot or, if indicated, a Leica TCS SP5 II confocal microscope and channel merging was performed using ImageJ software.