Dmi4000b inverted fluorescence microscope

Confocal imaging was performed with Zeiss LSM 780 confocal microscope. Light microscopy imaging was performed with Leica DM IL LED inverted microscope, and fluorescence images were collected with Leica DMI4000 B inverted fluorescence microscope. Image analysis was performed with ImageJ (v1.53o).

Cell sections were deparaffinized in xylene, and slides hydrated in 95% ethanol for 5 min, 85% ethanol for 5 min; slides were then hydrated in distilled water. Hematoxylin staining was performed for 3 min, and slides rinsed with distilled water for 2 min; 1% hydrochloric acid alcohol was used for 2 s to differentiate the stain. The sections were rinsed with tap water for 15 min followed by 1–2 s of distilled water. Slides were stained with eosin for 30 s. Differentiation was determined according to the color, and 80% ethanol was used to differentiate stains. Slides were further dehydrated with 85% ethanol for 5 min, followed by 95% ethanol for 5 min. Then, the slides were dehydrated with anhydrous ethanol for 10 min. After the run off was transparent, slides were sealed by adding a drop of neutral gum. Observation and photography were performed with a microscope (Dmi4000b inverted fluorescence microscope, Leica, Germany).

Cells were examined under a Leica DMI4000 B inverted fluorescence microscope using a 63x water objective. GFP was visualised with an excitation filter BP 470/40, beam splitter 500, and BP 525/50 barrier filter. Flow cytometry of cells expressing GFP-tagged Pdr11p was performed as described previously [13 (link)].

The GCPMs and primary myotube cells (GCPMTs) were fixed in 4% paraformaldehyde for 0.5 h, followed by three washes with PBS for 5 min each time. The cells were then permeabilized with 0.5% triton X-100 at room temperature for 0.5 h and washed three times with PBS for 5 min each. Subsequently, the samples were blocked with 5% goat serum for 0.5 h and finally incubated overnight at 4 °C with primary antibodies (Desmin diluted 1:100 and MYHC diluted 1:5). Following this, the cells were washed three times with PBS for 5 min each and incubated at room temperature for 1 h with a second antibody according to the type of secondary antibody (diluted at 1:1,000). Cells were finally stained with 4',6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature and covered with glass cover slides. Antibodies information is listed in Table S3. For the paraffin section of muscle tissue immunofluorescence staining, the 5 μmol/L slices were repaired with antigen repair solution (0.2 g citric acid + 1.5 g trisodium citrate diluted with 500 mL pure water), treated with 3% hydrogen peroxide, and then blocked. The blocking and subsequent steps consisted of the immune crown staining of cells. Immunofluorescence images were obtained using a DMI4000B inverted fluorescence microscope (Leica). The information on primary and secondary antibodies and DAPI is given in Table S1.

To detect EV uptake, EVs were first labeled with the lipophilic red fluorescent dioctadecyl (DiD) Vybrant stain (Invitrogen). A total of 100 μg of HEK293EVs and NK3.3EVs in 50 μL PBS were each labeled with 1 μL of 50 μM DiD stain for 20 min at 37 °C. After incubation, 450 μL PBS was added to the EVs, followed by 167 μL ExoQuick. Suspensions were mixed gently and incubated for 2 h at 4 °C. Tubes were centrifuged at 10,000 rpm for 10 min, supernatants aspirated, and EV pellets resuspended in 50 μL PBS and applied to cells. Monolayer and mammosphere cells were seeded as described above. After EV treatment, cells and spheres were gently washed to remove non-internalized EVs. Control cells were incubated with DiD alone. HFF and MDA-MB-231 monolayer cells and MCF7 mammospheres were imaged in situ with a Leica DMI4000B inverted fluorescence microscope. MCF7 U-bottom spheres were imaged in situ with the Leica inverted fluorescence microscope, a Bio-Rad ZOE fluorescent cell imager, and inverted phase microscope.
EV-treated HEK293 and GFP+ MCF7 spheres were removed from wells, washed with PBS, fixed in 100 μL 4% paraformaldehyde (PFA) for 30 min, and then transferred in PFA to covered glass chamber slides. Images were obtained by Leica spinning-disk or confocal fluorescence microscopes in the Research Microscopy and Histology Core, Saint Louis University.