Phalloidin atto 550

LX2 cells were seeded at a density of 3 × 10⁵ cells per well on coverslips. Cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X‐100 in PBS for 10 min, and counterstained with antibodies against vimentin (1 : 100, 2707‐1, Epitomics), Hif‐1α (1 : 100, ab16066; Abcam), P62 (1 : 100, PM045, MBL, USA), or Phalloidin‐Atto 550 (1 : 50, 19083, Sigma‐Aldrich). The coverslips were mounted onto slides in antifade mounting medium (P0126; Beyotime), and fluorescent images were captured (DMI3000B; Leica, Wetzlar, Germany).

Adult flies were narcotized with CO₂. After cutting off the head, the flies were pinned through the thorax with their ventral side facing up onto a wax dish. After covering the flies with PBT the abdomen was opened along the ventral side. Next the gut was removed from the abdomen using forceps, transferred into a staining dish, and fixed for 20 – 40 min in PBS containing 3,7% formaldehyde. Guts were stained over night at 4° with Phalloidin-Atto-550 (1:3000; Sigma-Aldrich), washed three times with PBS and embedded in Vectashield (Vector Laboratories).
For the analysis of the wing bristles adult flies were narcotized with CO_2, the wings were removed at the notum using forceps and embedded in Euparal (Roth).

HeLa T-REx cells expressing fluorescently labeled fusion proteins were grown on glass cover slips. After paraformaldehyde (3.7%) fixation cells were stained with DRAQ5 (Biostatus, UK) and Phalloidin-Atto 550 (Sigma, St. Louis, USA) to label nuclei and actin, respectively. Cover slips were mounted with ImmuMount (Thermo Scientific, Pittsburgh, PA) as has been described previously55 (link). Fluorescence images of adherent cells were generated with a Zeiss LSM 710 confocal microscope (Zeiss, Jena, Germany), using the Zeiss LD C-apochromat 40x/1.1 water objective. Images represent confocal slices of approximately 1 μm and were analyzed with the ZEN 2012 software (Zeiss, Jena, Germany).
Information on statistics and sample numbers is given in the corresponding figure legends.

Samples were fixed using 4% paraformaldehyde solution. The fixation was quenched with 25 mM NH₄Cl/PBS. In case of 3D samples, scaffolds were incubated in 5% gelatin, 5% sucrose dissolved in PBS at 37°C with subsequent solidification at 4°C to enable cutting of cylindrical samples into halves. Cylinder halves were embedded into Tissue‐Tek* O.C.T. Compound (#25608‐930, Sakura Inc.) and planed using a CryoStat (LEICA CM3050S). Samples were rinsed thoroughly in PBS at 37°C to remove gelatin/sucrose and Tissue‐Tek, respectively. The following antibodies were used: mouse‐anti‐vinculin (#V9131, Sigma Aldrich), FLAG (740001, Thermo Fischer) and goat‐anti‐mouse IgG‐488 (#A‐11029, Thermo Fischer). F‐actin and cell nuclei were visualized using SYTOX™ Green (#S7020, Thermo Fischer), Draq5 (#424101, Biolegend) and Phalloidin‐Atto550 (19083, Sigma Aldrich).

Actin filaments were stained with phalloidin to define HL-1 cell area (73 (link)). Briefly, the cell monolayer was washed twice with PBS for 5 min, fixed with 4% (w/v) paraformaldehyde (PFA) in PBS (Sigma) for 15 min and permeabilized with 0.1% (v/v) TritonX-100 (Sigma) in PBS twice for 2 min, at room temperature; PBS washes (2 × 5 min) were performed after each step. Cells were incubated with Phalloidin-Atto 550 (1:300; Sigma) in PBS for 1 h at room temperature to stain cytoskeletal F-actin. 4′,6-Diamidino-2-phenylindole dihydrochloride solution (DAPI; 1:1,000, Sigma) was also added to the solution to stain the nuclei. Excess dye was removed by PBS wash (2 × 5 min) and the slide was mounted with Aqua-Poly/Mount aqueous mounting medium (Polysciences, Inc.). Images were acquired at a fluorescence optical microscope (DM 2500, Leica Microsystems, Germany) (20× magnification). The cell area was measured manually with ImageJ software¹ and expressed as pixels.

For confocal microscopy experiments, the cells were seeded on glass coverslips and let adhere for 24 hours prior to the double exposure. At the end of the exposure period, the cells were washed twice for 5 min in PBS, fixed in 4% paraformaldehyde for 30 min at room temperature. After two washes (5 min in PBS), they were permeabilized in Triton X100 (0.1% w/v) for 5 min at room temperature. After two more washes in PBS, Phalloidin-Atto 550 (Sigma) (200 nM) or Phalloidin-Atto 390 (500 nM) was added to the cells and let for 20 min at room temperature in the dark. Coverslips-attached cells were washed, and cell nuclei were colored via DAPI for 5 minutes at room temperature, protected from light (1µg/ml, Eurobio, Les Ulis, France). The coverslips were washed twice and placed on microscope slides (Thermo Scientific, Illkirch, France) using a Vectashield mounting medium and imaged using a Zeiss LSM 880 confocal microscope. The images were processed using the ImageJ software.

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X and blocked with 5% bovine serum albumin (BSA) for 1 h. Samples were incubated overnight at 4 °C with the primary antibodies CD44 (BD Pharmingen), CD29 (R&D Systems), CD90.2 (BD Pharmingen), CD31 (Abcam, Cambridge, UK), MyHC (in house production), Pax7 (DSHB, Iowa, USA), and MyoD (Santa Cruz Biotechnology, Heidelberg, Germany). Primary antibodies were detected using goat anti-rabbit or goat anti-mouse IgG antibodies conjugated to fluorescein isothiocyanate (FITC) or Streptavidin Alexa 594. Cells were counterstained for F-actin with phalloidin-FITC (Sigma-Aldrich) or phalloidin-Atto 550 and for nuclear staining with DAPI (Sigma-Aldrich).
Cells were visualized using a fluorescence microscope (Nikon Eclipse 800) or confocal laser scanning microscope (Zeiss LSM 710; Carl Zeiss Microscopy, Oberkochen, Germany).
For fiber formation assay the cultured myofibers were fixed with methanol for 7 min and stained with Giemsa solution (1:20) for 1 h at room temperature. The number of myofibers per high-power field was calculated along with the number of nuclei per myofiber.