Tissue tek oct compound

For the detection of epidermal innervation, skin samples were processed as described before (Arcourt et al., 2017 (link)). Skins from hind paw footpad of P0 pups were dissected, fixed in methanol/acetone (1:1) for 30 min at -20°C, and then cryoprotected in 30% sucrose overnight at 4°C. Samples were embedded in Tissue-Tek O.C.T compound (Electron Microscopy Sciences, Hatfield, PA, United States) and sectioned into 45 μm. Sections were incubated in 50 mM Glycine for 45 min at room temperature, blocked in 10% goat serum, 1% BSA, and 0.5% Triton X in PBS for 1 h at room temperature, and incubated with rabbit anti-PGP9.5 (Proteintech Group, Inc, Rosemont, IL, United States) with a dilution of 1/300 to 1/500 overnight at 4°C. Sections were then incubated with anti-rabbit Alexa Fluor 594 (Invitrogen, Carlsbad, CA, United States) with a dilution of 1:500 at room temperature for 1 h and counterstained with Hoechst for nuclei for 5 min at room temperature. Images were acquired by confocal microscopy (Leica TCS SP8 Confocal Microscope). For each mouse, 10–15 sections throughout the section thickness (45 μm.) and entire length (200 μm) were analyzed to identify all of the fibers and the intensity of PGP9.5-labeled fibers in the epidermis region was quantified using ImageJ.

Organs were embedded in Tissue-Tek O.C.T Compound (Electron Microscopy Science) and frozen in liquid nitrogen. Six to 8 μm sections were cut, fixed in cold acetone and then blocked with 2% BSA. The following antibodies were used: mCLCA1 (hamster mAb 10.1.1), Lyve-1-A488 (ALY7, eBioscience), ITGA2b-APC (MWReg30, Biolegend), Prox-1 (polyclonal goat, R&D Systems), GPIbß (RAM1-A645), MAdCAM-1 (MECA-367, BD-Pharmingen), fibronectin (Rabbit polyclonal, Patricia Simon-Assmann, UMR-S 1109 INSERM, Strasbourg), RANKL (IK22.5, Rat IgG2a, [22 (link)]), goat anti-rabbit-A488 (Molecular Probes), goat anti-hamster-A488/A546 (Molecular Probes), donkey anti-rat-Cy3 (Jackson) or streptavidin A546 or A647 (Molecular Probes). Sections were mounted using DAKO mounting medium (Dako, Hamburg, Germany). Images were acquired and treated as noted above.

Following the imaging session, rats were euthanized and lungs were harvested. They were frozen in OCT (Tissue-Tek™O.C.T. Compound, Electron Microscopy Sciences, USA) and stored at −80 °C shielded from light. Lung sections were cut and stained with DAPI ((4′,6-Diamidino-2-Phenylindole, Dihydrochloride), ThermoFisher Scientific, USA) in order to detect cell nuclei and fluorescent DiD-MSCs. Images were acquired on a fluorescence microscope (AxioVert A1, Zeiss, Germany) with filters mPlum for DiD and DAPI and magnification 20X.

Animals were mounted ventral up in Vectashield mounting medium. Fluorescent images were taken with a Zeiss LSM700 Confocal Microscope using ZEN 2011 software with following objectives. For transverse cryosection shown in Fig. 4e and Supplementary Fig. 9b, stained animals were embedded in Tissue-Tek O.C.T. Compound (Electron Microscopy Services) and sectioned at 25 μm. All images and quantification for co-expression and for determining cell localization relative to muscle fiber layer were analyzed at 40X NA 0.75 with minimum 0.31 × 0.31 × 1.11 μm resolution or 63X NA 1.4 with 0.2 × 0.2 × 0.57 μm resolution with the exception of H3P⁺smedwi-1⁺ (Fig. 4d) which was quantified at 20X NA 0.8 with minimum 0.63 × 0.63 × 2.04 μm resolution. Animal overview images were acquired with 10X NA 1.45 with minimum 1.25 × 1.25 × 7.26 μm resolution. Linear adjustments of brightness and contrast equally across controls and experimental images, re-assignment of linear look-up table colors, co-localization analysis of FISH signals, and maximum intensity projections through area of interest was performed using Fiji with ImageJ 2.0.0^{76 (link)}. Live animal images were taken with a Zeiss Discovery Microscope using AxioVision v4.7.1.