Pap pen

Adult ventricular cardiomyocytes were infected at an MOI of 5 as for live imaging and kept in storage solution in a humidified incubator at 37°C, 5% CO2. At 48 hours an aliquot was taken and paraformaldehyde to a final concentration of 4% was added, at 10 minutes, cells were pelleted (200rpm, 2min) in a benchtop centrifuge, washed with 1xPBS and resuspended in PBS. Cells were spun (Cytospin, Thermo Scientific) onto glass slides, and ringed with a PAP pen (Sigma). Permeabilisation with 0.1% Triton-X-100 in Tris Buffered saline for 10 minutes at room temperature was followed by blocking (0.2% albumin in permeabilisation buffer) for 20 minutes. Primary antibodies (mouse monoclonal 9E10 anti-myc (Santa-Cruz), and rabbit polyclonal anti-DsRed (Clontech) were diluted 1:200 in blocking buffer. Three hours after primary incubation cells were washed in permeabilisation buffer, and counter stained with Alexa-488 anti-mouse, and Alexa-568 anti-rabbit fab fragment secondaries (Invitrogen), nuclear counterstaining was with Topro3 (Invitrogen), for an hour before washing and mounting (Vectashield, Vector labs). Images were acquired on a Leica SP5 confocal microscope with a 63x oil immersion lens. hSC-CM’s were plated onto 0 thickness coverglass, infected at an MOI of 5. Cells were fixed and stained 48 hours after infection as above.

In order to assess cellular morphology, immediately after functional testing engineered muscles from normoxic and hypoxic conditions were fixed by the drop wise addition of ice‐cold methanol‐acetone solution. Subsequently constructs were removed from their sutures and adhered to poly‐L‐lysine coated microscope slides and ringed with PAP pen (Sigma–Aldrich) before being permeablized with 1× Tris buffered saline (TBS: 0.5M) and 0.2% Triton x‐100 (Fisher Scientific, UK) for 2 h. Constructs were then incubated in rhodamine conjugated phalloidin (Fisher Scientific) diluted 1:200 in TBS to label F‐actin, and DAPI (Sigma–Aldrich) diluted 1:1000 in order to label cellular nuclei, and were incubated at room temperature in the dark for 3 h. Following 4 washes in distilled water, constructs were mounted on to glass coverslips using a drop of Fluoromount™ medium (Sigma–Aldrich). Images were captured using a Leica DM2500 Fluorescent microscope at 40 × magnifications and analysis was conducted using Image J software (NIH, USA), with a minimum of five images and 40 myotubes analyzed per engineered muscle.

EdU-labeling of hemocytes was performed using the Click-iT EdU Alexa Fluor 647 Imaging Kit (Invitrogen, Molecular Probes #C10356). Hemocytes from third instar larvae were dissected into 15 μl of 10 μM EdU and allowed to settle for 30 min into 5 mm wells on Superfrost Plus microscope slides made with a pap pen (Sigma-Aldrich #Z377821). After 30 min of EdU incorporation, the solution was removed and replaced with 4% paraformaldehyde for 10 min. The samples were washed twice with PBS-T. Samples were incubated with primary mouse anti-P1 and fluorescent secondary antibodies as described above. The cycloaddition reaction was performed per the manufacturer’s instructions. Samples were mounted in Vectashield. We imaged 10–15 areas of the slide at random at 25× using a Zeiss LSM510 confocal microscope. To calculate the percentage of proliferating plasmatocytes, we used ImageJ to quantify the number of P1-positive and EdU-positive cells.

Cross-sections of the lumbar enlargement, 12 μm thick, were obtained using a cryostat (Microm, HM525), transferred to gelatin-coated glass slides, and stored at -20 °C until analysis. To perform immunohistochemistry, the slides were brought to room temperature, and the sections were delineated with a hydrophobic pen (PAP pen, Sigma Z377821). Subsequently, the slides were placed in a humidity-controlled chamber, protected from light. Sections were immersed in 0.01 M PBS (3 × 5 min each, pH 7.38), and incubated in 150 µL of blocking solution (3% bovine serum albumin in 0.1 M PBS, pH 7.38) for 45 min. Following this step, the primary antibodies were diluted in an incubation solution (1.5% bovine serum albumin and 0.2% Tween in 0.1 M PBS, pH 7.38) overnight at 4 °C.
After incubation with the primary antibody, sections were washed with 0.01 M PBS and incubated at room temperature with the appropriate secondary antibody for 45 min. The sections were then washed with 0.01 M PBS, dried, and cover-slipped using a glycerin/PBS (3:1) mounting medium. The slides were examined using an epifluorescence microscope (Leica DMB5500) and documented with a digital camera (Leica DFC 345 FX) equipped with specific filters, depending on the secondary antibodies used. The antibodies used are detailed in the Supplementary Table 1.