Polysine slides

Muscles were mounted onto cork with OCT and 8 μm fresh frozen sections were cut and collected onto poly-sine slides (VWR). In the case of the heart, the sections were cut transversely at the level of the papillary muscles for the first study. In the second study sections were taken at 6 different levels. For the TA and QUAD, transverse sections were collected approximately mid-way along the muscle. Hemi-diaphragm pieces were rolled and mounted such that transverse sections could be cut though the full width.

DT40 cells were grown in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Labtech), 1% chicken serum (Gibco) and 100 U µl⁻¹ penicillin/streptomycin at 39°C, 5% CO₂. To arrest cells in mitosis, cultures were treated for 12–14 h with 0.5 µg ml⁻¹ nocodazole (Sigma-Aldrich). For SILAC labelling cells were grown in RPMI media (Invitrogen) with 10% (v/v) dialysed FBS (Sigma), 100 µg ml⁻¹ U-13C615N2-l-lysine : 2HCl and 30 µg ml⁻¹ U-13C615N4-l-arginine : HCl (Sigma) for six cell cycles.
To measure the mitotic index, cells were adhered to Polysine slides (VWR). Slides were incubated in 75 mM KCl for 4 min at room temperature and fixed in 75% methanol/25% acetic acid. The slides were inspected under a light microscope and 1000 cells were counted for each condition.

Mice were killed by cervical dislocation. Muscles were immediately dissected and divided into two. One half was snap-frozen in liquid nitrogen for chemical analysis and the other in liquid nitrogen cooled isopentane (Sigma) mounted on 20 mm cork discs with OCT embedding matrix (both Raymond Lamb, Loughborough, UK) for sectioning. 10 µm sections for light microscopy were cut on a Bright Clinicut microtome at −20°C and mounted onto polysine™ slides (VWR International, Leuven, Belgium) and allowed to air dry. Samples were stored at −80°C and slides at −20°C.
For protein quantification, frozen tissue was powdered using a chilled pestle and mortar under liquid nitrogen, suspended in ice-cold lysis buffer (975 µl HEPES, 10 µl Protease Inhibitor Cocktail, 10 µl PMSF, 5 µl Na₃Va₄), and centrifuged at 5000 g for 20 min at 4°C. Samples were assayed in duplicate. Mouse and human VEGF protein (R&D Systems, Abingdon, UK), and PGF1α (Cambridge Biosciences, UK) was measured by enzyme linked immune sorbent assay (ELISA), according to manufacturer's instructions (5 animals/group). Total tissue protein concentration was determined by Bradford assay (using Bradford reagent, Sigma) performed in duplicate at the same time as the ELISA.

Pancreata and graft-bearing pinnae were fixed in 4% PFA for 72 h, dehydrated in sucrose, and mounted in OCT. 10 µm sections were cut on a Leica cryostat and placed on Polysine slides (VWR), air dried, and fixed again for 10 min in acetone. Sections were stained using guinea pig-anti-insulin antibody (DAKO) at a 1:40 dilution and detected with anti-guinea pig Alexa 546 (Molecular Probes). Rabbit anti-human glucagon (Millipore) was used at a 1:50 dilution and detected with anti-rabbit Alexa 488 (Molecular Probes). Anti-mouse CD4 (BD) was detected with anti-rat Alexa 488 (Molecular Probes). Nuclei were visualized with DAPI (Molecular Probes). The sections were viewed using either a Zeiss Axioskop 2 or LSM700 Confocal (Zeiss) and processed using Zen software.

Muscle samples were freed from blood and non-muscle material, embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude, The Netherlands), frozen in liquid nitrogen-cooled isopentane and stored in liquid nitrogen until further processing. For immunohistochemical staining procedures, 7 µm cross-sections of all biopsy time points were mounted in double on Polysine^® slides (VWR International GmbH, Darmstadt, Germany), carefully aligned for cross-sectional analysis, air-dried and stored at −80 °C. Immunohistochemical procedures were conducted according to previous works [21 (link),54 (link)]. Skeletal muscle cross-sections were incubated overnight at 4 °C with specific primary antibodies recognizing SUMO-1 (#4930; Cell Signaling, Beverly, MA, USA), nuclear envelope marker lamin-A (ab26300, Abcam, Cambridge, UK) and A 4.951, which is raised against adult human slow myosin heavy chain (MyHC1) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Antibodies were diluted 1:150 (SUMO-1), 1:200 (lamin-A) or 1:200 (A4.951) in TBS containing 1% bovine serum albumin (BSA). Muscle cross-sections from all biopsy time points of each subject were stained within a single batch to minimize variability in staining efficiency. Double-mounted, consecutive serial sections on the same slide were stained simultaneously for slow MyHC1 and SUMO-1.

To demonstrate myelin compaction, myelinoids were fixed in phosphate buffer (PB) containing 4 % PFA and 2 % glutaraldehyde (Agar Scientific) for 2 hours, post-fixed in 0.1 % glutaraldehyde for 24 hours at 4 °C and washed in 0.1 M PB (2 x 15 min). Samples were transferred to 1 % OsO₄ in 0.1 M PB for 45 min at RT, washed in 0.1 M PB and then dehydrated in increasing concentrations of EtOH for 15 minutes each (50 %, 70 %, 90 %, 100 % x 3). The Epoxy Resin (Araldite) kit (Agar Scientific) was used for embedding samples. Briefly, samples were incubated in a 1:1 mix of Araldite-DDSA:acetone for 1 hour then transferred to Araldite-DDSA for overnight incubation before being cured in Araldite embedding mix at 60 °C for 48 hours. For toluidine blue staining, 1 μm semithin sections were cut, placed on Polysine slides (VWR) and flattened by rotating the slide on a heat plate. The slide was then flooded with toluidine blue (Wako, 5% in a Borax solution) and heated again until vapours evolved. The slides were washed in water, mounted with FluorSave and imaged on a Zeiss Observer microscope. For Transmission electron microscopy, ultrathin sections, 60 nm thick, were cut, stained in Uranyl Acetate and Lead Citrate then viewed using a JEOL JEM-1400 Plus TEM. Images were captured on a GATAN OneView camera.