Cryoprotective Agents

An 8-day-old ‘wildtype’ C56BL/J mouse was euthanised in a schedule 1 procedure via intraperitoneal injection of sodium pentobarbital followed by decapitation following licensed procedures approved by the Mary Lyon Centre and the Home Office UK as described for the brain tissue. Heart was dissected and placed in ice-cold Millonig’s buffer (Fisher Scientific, Thermo Fisher Scientific) then fixed using ice-cold 4% PFA in Millonig’s buffer (Fisher Scientific) then left to incubate overnight at 4°C. Thin sections of 50 µm were obtained using a vibratome and placed onto a glow discharged electron microscopy grid (UltrAuFoil, 200 Au mesh, 2/2 Au film; Quantifoil Micro Tools) that had been pre-clipped into Autogrids (Thermo Fisher Scientific). The sample was assembled in the mid-plate between two 6 mm planchettes incubated with hexadecane for 15 min. Twenty percent w/v BSA (Sigma-Aldrich, St Louis, MO, USA) in PBS buffer was used as a cryoprotectant and filler and the assembly was high-pressure frozen in a Leica HPM 100 (Leica Microsystems). The planchette-autogrid assembly was disassembled and stored under liquid nitrogen.

Following ex-vivo μCT, bones were placed in ascending sugar solutions as cryoprotectant (10%, 20%, 30%) at 4 °C for 24 h each, then cryo-embedded in SCEM medium (Sectionlab, Japan) and stored at −80 °C. Consecutive sections of 7 μm were prepared using a cryotome (Leica, Wetzlar, Germany) and cryotape (Cryofilm 2C(9), Sectionlab, Japan). Sections were fixed onto glass slides, air-dried, and stored at −80 °C until staining. Movat’s pentachrome staining comprised the following steps: sections were air dried for 15 min, fixed with 4% PFA (30 min; Electron Microscopy Sciences, Hatfield, USA), pretreated with 3% acetic acid for 3 min, stained 30 min in 1% alcian blue pH 2.5, followed by washing in 3% acetic acid under light microscopic control. Sections were rinsed in H₂O_dest and immersed in alkaline ethanol for 60 min, then washed in tap water followed by incubation in Weigert’s hematoxylin for 15 min. After washing in tap water for 10 min, sections were stained in crocein scarlet-acid fuchsin for 15 min, treated with 0.5% acetic acid for 1 min, followed by 20 min incubation in 5% phosphotungstic acid, and 1 min in 0.5% acetic acid. The sections were washed three times for 2 min in 100% ethanol, followed by incubation in alcoholic Saffron du Gâtinais for 60 min. The slides were dehydrated in 100% ethanol, cleared shortly in xylene, covered with Vitro-Clud and a cover slip. Imaging was performed on a Leica light microscope using LAS X software (Leica Microsystems GmbH, Wetzlar, Germany) at 10× magnification. Quantitative analyses of the Movat’s pentachrome staining were evaluated using an ImageJ macro. All analyses were performed blinded to sex, fixation, and pain management protocol.
Immunofluorescence staining was performed as described previously^{66 (link),67 (link)} using the following antibody: Endomucin (Emcn) (V.7C7 unconjugated, rat monoclonal, sc-65495, 1:100; Santa Cruz Biotechnology, Dallas, USA), goat anti-rat A647 (1:500; A-21247, polyclonal, Invitrogen, Thermo Fisher Scientific, Waltham, USA) and DAPI (1:1,000; Thermo Fisher Scientific, Waltham, USA). Blocking was performed with 10% FCS/PBS and the staining solution contained 5% FCS and 0.1% Tween20 (Sigma Aldrich, St. Louis, USA). Images were acquired using a Keyence BZ9000 microscope (Keyence, Osaka, Japan). The images were processed and analyzed with ImageJ^{69 ,70 (link)}. An area of interest was established and managed via the built-in ROI-Manager, while cell number and signal distribution within the area were determined using the plug-ins Cell-counter and Calculator Plus. Data was processed with the ImageJ plugin OriginPro.