Pap pen

Phi29 DNA polymerase was purchased from MBI Fermentas. CodeLink Activated Slides were purchased from SurModics Inc. (USA), and Vectashield was from Vector Laboratories Inc. (USA). CD44 MicroBeads (human), FcR Blocking Reagent (human), CD44-PE Antibody (human), CD133-PE Antibody (human), Casein Kinase II (CK2) and alkaline phosphatase from calf intestine were purchased from New England BioLabs Ltd. (UK). Pap Pen was purchased from Dako (Glostrup, Denmark).
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) reagent (M5655) was purchased from Sigma-Aldrich ApS (Denmark), dissolved in phenol red MEM at a final concentration of 5 mg/ml, sterilized using 0.22 µm filter and stored at −20°C.

Following 5 days of treatment, each engineered muscle was washed with PBS and fixed using ice cold methanol; acetone solution. Subsequently, engineered muscle constructs were cut away from the sutures and placed on poly‐lysine coated microscope slides (VWR, Leicestershire, UK) and ringed with PAP pen (DAKO, Cambridgeshire, UK). Constructs were blocked with 1× Tris buffered saline (TBS; 0.5 M) containing 5% goat serum (Sigma–Aldrich) and 0.2% Triton‐x‐100 (Fisher Scientific) for 90 min. Following three washes with TBS, constructs were incubated overnight in a humidified staining chamber with rabbit polyclonal anti‐desmin primary antibody (Abcam, Cambridgeshire, UK) diluted 1:200 in TBS. After overnight incubation, constructs were washed three times in TBS and incubated for 3 hr with goat anti‐rabbit TRITC secondary antibody (Abcam) diluted 1:200 in TBS, and DAPI (Sigma–Aldrich) in order to visualize nuclei. Following three further washes in distilled water, constructs were mounted on glass coverslips using a drop of Fluoromount™ (Sigma–Aldrich) mounting medium. Engineered muscle constructs were imaged using a Zeiss LSM‐710 confocal microscope (Zeiss, Cambridgeshire, UK) and were analyzed using Image J software (NIH).

Hybridization was performed using the probe EUB338 labeled with Cy3, whose sequence is complementary to the sequence of a region within the 16S rRNA that is common to all eubacteria as well as the mixture probe, EUB338 I, II, III. The complementary probe non-EUB338 labeled with Cy3 was used as control. Tissue sections were prepared using sterile microtome equipment and deparaffinized in coplin jars by two changes of xylene and dehydrated twice with 99.9% ethanol for 3 min at each step. Slides were air dried and a circle was drawn around the tissue section using a hydrophobic pen (Dako PAP pen; Glostrup, Denmark). The hybridization and washing steps were performed as previously reported [22] . Fixed samples were hybridized by the application of 10 ml of hybridization buffer containing 5 ng of each specific oligonucleotide probe at 45°C for 90 min. Stringent washing was performed by incubating the slide in washing buffer (20 mM Tris-HCl pH 7.6, 0.01% sodium dodecyl sulfate, 112 mM NaCl) at 48°C for 15 min. Finally, the slides were rinsed with water, stained with DAPI, air dried, and mounted in Citifluor (Citifluor Ltd., London, United Kingdom). Slides were examined using a Nikon fluorescent microscope equipped with a standard filter set (Nikon, Germany).