Dpx mounting medium

Muscle sections were briefly incubated for 30 minutes at 37°C in a sodium phosphate buffer containing 75mM sodium succinate (Sigma), 1.1mM nitroblue tetrazolium (Sigma) and 1.03mM phenazine methosulphate (Sigma). Samples were then fixed in 10% formol-calcium and cleared in xylene prior to mounting with DPX mounting medium (Fisher). Photographic quantification of the samples was performed on a Zeiss Axioskop2 microscope mounted with an Axiocam HRc camera. Axiovision Rel. 4.8 software was used to capture the images.

Transverse EDL muscle sections were incubated for 3 min at room temperature in a sodium phosphate buffer containing 75 mM sodium succinate (Sigma), 1.1 mM nitroblue tetrazolium (Sigma) and 1.03 mM phenazine methosulphate (Sigma). Samples were then fixed in 10 % formal-calcium and cleared in xylene prior to mounting with DPX mounting medium (Fisher). Photographic quantification of the samples was performed on a Zeiss Axioskop2 microscope mounted with an Axiocam HRc camera. Axiovision Rel. 4.8 software was used to capture the images.

In situ hybridization was performed as previously described^{45 (link)}. Briefly, paraffin-embedded sections were dewaxed and rehydrated in PBS, post-fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilized with proteinase K (10 μg/ml for 15 min, Roche), re-fixed in 4% PFA for 5 minutes and treated with 0.25% acetic anhydride for 10 min prior to hybridization with DIG-labelled probes (1:100 in hybridization buffer; Amresco) overnight at 65 °C. Sections were washed twice at 65 °C for 30 min (1 × SSC, 50% formamide, 0.1% Tween 20) followed by treatment with 20 μg/ml RNase A (Roche) for 30 min at 37 °C. Slides were then incubated with the anti-DIG antibody (1:2500, Roche) overnight at 4 °C. Probes were detected using NBT/BCIP (Promega) according to the manufacturer’s instructions. Sections were counterstained with Nuclear Fast Red (Sigma-Aldrich), dehydrated and cleared in xylene and mounted in DPX mounting medium (Ajax Finechem). Slides were imaged using a Mirax digital slide scanner (Carl Zeiss) with a 20X objective.

Acetonitrile and methanol both HPLC grade, DPX mounting medium, xylene and Optiphase scintisafe gel were purchased from Fischer Scientific (Leicester, UK). Tetracaine base BP grade (99.9%), formalin solution neutral buffer 10%, DAPI medium, ethanol, heparin sodium salt (I-A), urethane, 0.7% glacial acetic acid, isopropanol and FTIC-dextran with average molecular weight (Mw) of 4 kDa (FD-4) and 10 kDa (FD-10S) used without any further purification steps were supplied by Sigma Aldrich (Dorset, UK). Concentrated hydrochloric acid and sodium hydroxide was from Fluka (Dorset, UK). Sodium acetate was provided by Alfa Aesar (Heysham, UK). Silicone membranes with a thickness of 0.25 mm were purchased from GBUK Healthcare (Selby, UK). Phosphate buffered saline (Dulbecco A) tablets were obtained from Oxiod Limited (Hampshire, England). The Tissue-Tek® O.C.T™ compound, scintillation vials and hydrogen peroxide 30% were obtained from VWR International (Leicestershire, UK). Dextran (carboxyl-¹⁴C) with an average M.W. of 10 kDa and specific activity of 0.00006 Ci/mmol was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, USA). Soluene® 350 was provided by Perkin Elmer (Bucks, UK). Isoflurane 100% (w/v) inhalation vapour liquid was obtained from Animal Care Ltd. (York, UK).