Goat serum

To detect GFP, phospho-Paxillin, Vinculin or Myogenin, larvae were anesthesized in 0.02% MS-222, fixed in 4% paraformaldehyde (PFA, Sigma) overnight at 4°C and subsequently stored in 100% methanol (Fisher) at −20°C. Samples were washed in 0.1% Tween20 in phosphate-buffered saline (PBT, Sigma) and permeabilized. Samples were blocked in 5% goat serum (Life Technologies) in PBT for at least 2 h, then incubated with primary antibody diluted in 5% goat serum/PBT over night at 4°C. Samples were washed for at least 4 h in 0.1% PBT the following day, then incubated with secondary antibodies diluted in 5% goat serum/PBT for 2 h at room temperature. After several washes in PBT, samples were taken through glycerol series and mounted in VectaShield with DAPI (Vector Laboratories).
To label dividing cells with BrdU, larvae were exposed to 10 mM BrdU diluted into E3 medium and processed as previously described [16 (link)].
Antibodies used were chicken polyclonal anti-GFP (1 : 500; Millipore), rabbit polyclonal anti-GFP (1 : 500; Life Technologies), rabbit polyclonal anti-phospho-Paxillin (Tyr118) (1 : 200; Thermo Scientific), mouse anti-Vinculin (1 : 200; Sigma), rabbit polyclonal anti-Myogenin (1 : 50; Santa Cruz Biotechnology), rat monoclonal anti-BrdU (1 : 250; Abcam). Secondary antibodies used were Alexa conjugated antibodies diluted 1 : 500 in goat serum and PBT.

To measure the thickness of self-assembled scaffold, expression of collagen type I in extracellular matrix produced by ASCs was detected. Hence, self-assembled scaffold was fixed in 4% (w/v) paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MO, USA) for 20 min at 4 °C followed by three times washing with phosphate buffer saline (PBS)(1×) (Invitrogen, Waltham, MA, USA). Cell permeabilization was performed with 0.5% (v/v) triton-X (Sigma-Aldrich, St. Louis, MO, USA) with 20 min incubation at room temperature followed by three times washing with PBS (1×). Nonspecific binding was blocked using 10% (v/v) goat serum (Thermo Fisher, Waltham, MA, USA) at 37 °C for 1 h. Mouse monoclonal anti-collagen I antibody (COL-1/ab6308, Abcam, Cambridge, UK) with 1:200 dilution ration in 1% (v/v) goat serum was used as primary antibody at 4°C for overnight incubation in the dark. Cells then were washed three times with PBS (1×) followed by incubation with secondary antibody, goat anti-mouse Alexa Fluor 594 (Invitrogen, Waltham, MA, USA) with 1:300 dilution ration in 1% (v/v) goat serum at 37 °C for 2 h. The cells then were washed three times with PBS (1×) and were counterstained with 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Life Technologies, Carlsbad, CA, USA) diluted in PBS (1×) with 1:15,000 dilution ratio at room temperature for 20 min.

Cells were fixed in the PDMS chambers for 20 min with 4% paraformaldehyde, blocked with 10% donkey serum (Merck Millipore, Germany) or 10% goat serum (Life Technologies), and permeabilized with 0.1% Triton-X. The PDMS substrate was cut into smaller pieces to be incubated overnight at 4 °C with primary antibodies diluted in 1% donkey serum or 1% goat serum (Supplementary Table 1). This was followed by incubation with appropriate secondary antibodies for 1 hr. The secondary antibodies that were used included goat anti-rabbit Alexa Fluor 488, goat anti-mouse Alexa Fluor 546 (Life Technologies), or donkey anti-goat 488 (Abcam, England, UK) at the concentration of 1:500. Cells were counterstained with DAPI (Sigma-Aldrich) for 20 min and mounted with Prolong Gold antifade mounting media (Life Technologies). Cell images were taken with Leica DM IRM inverted microscope (Leica, Germany) or confocal microscope (Zeiss LSM710, Germany).

Cells were rinsed with PBS(−/−), fixed with 4% paraformaldehyde (Alfa Aesar) for 20 min at room temperature, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS (PBSX) for 10 min, blocked with 5% goat serum (Life Technologies, #50062Z) in PBSX for 1 h at room temperature, and incubated with antibody diluted in blocking buffer (5% goat serum in PBSX) overnight at 4 °C, followed by incubation with fluorescent-conjugated secondary antibody for 1 h at room temperature; nuclei were stained with DAPI (Invitrogen) after secondary antibody staining. Cell apoptosis/ necrosis assay kit (Abcam, #ab176749) was used to quantification cell death according to manufacturer’s instructions. Immunostaining of Ki67 (Abcam, # ab15580) was used to measure cell proliferation. Fluorescence imaging was conducted using a confocal laser-scanning microscope (SP5 X MP DMI-6000, Germany) and image processing was done using the ImageJ software (version 2.1.0). DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride) was purchased from Invitrogen (#D1306). The list of antibodies used for immunostaining can be found in the antibody list (Supplementary Table 2).