Paraformaldehyde (pfa)

The hydrated bone sample was placed into a fixation buffer of 0.8% paraformaldehyde (prepared from the powder form, Acros Organics, New Jersey, USA) and 0.2% glutaraldehyde (Sigma-Aldrich, St. Louis, Missouri, USA) for 24 h.^{64 (link)} After the cross-linking process, an X-ray image (Faxitron microradiography system; Faxitron, Tucson, Arizona, USA) with the setting of 25 KV, 6 s of exposure, and 3X distance of the cross-linked bone was acquired to establish a baseline mineralization level. The cross-linked sample was immersed in a mixture of 20% EDTA solution (Fisher Chemical, New Jersey, USA), 0.2% paraformaldehyde and 0.05% glutaraldehyde^{64 (link)} on a shaker for over 30 days with solution changes every 3 days.
Full demineralization was confirmed by X-ray image. A micro-CT image (In Vivo Bruker/Skyscan 1176; Bruker, Billerica, Massachusetts, USA) with the setting of 9 μm resolution, 0.5 mm aluminum filter, 2 frame averaging, and 0.3 degree rotation step was independently taken for the demineralized sample, which was verified by the X-ray image, to ensure that the sample was indeed fully demineralized. The fully demineralized sample was dehydrated in a vacuum oven (80 °C, overnight) before PALS measurement. The mass before and after demineralization and dehydration was measured to calculate the mass fraction of mineralization loss.

Mice were euthanized with an overdose of pentobarbital (50 mg/kg, i.p., Cat#: 2821, Vortech, USA) or CO₂ inhalation. After washing out blood, blood vessels and the other organs were dissected after perfusion fixation with 4% paraformaldehyde (Cat#: J19943-K2, Thermo Fisher Scientific, USA) for 5 min and further fixed with 4% paraformaldehyde in 4°C overnight, and then embedded using Optimal cutting temperature (O.C.T.) compound (Cat#: 4585, Fisher Health Care, USA) prior to cryosection. The frozen tissue sections with a 5-µm thickness were subject to immunochemical staining and morphological analyses as described elsewhere.^{25 (link)} The antibodies used are listed in Online supplementary Tables S1 and S2.

Ciliary ganglia (CG) from seven adult or young adult, male macaque monkeys (Macaca fascicularis), that had been sacrificed in the context of other research projects, were investigated in this study. At the time of sacrifice, the animals were sedated with a dose of ketamine hydrochloride (10 mg/kg, i.m.) and deeply anesthetized with sodium pentobarbital (100 mg/kg, i.v.). Next, animals were transcardially perfused with 0.1 M, phosphate buffered saline (PBS) (pH 7.2), followed by a fixative containing 4% paraformaldehyde in 0.1 M, phosphate buffer (PB) (pH 7.4) for light microscopic examination (4 animals) or a mixture of 1.0% paraformaldehyde (Fisher Chemical, Pittsburgh, PA) and 1.5% glutaraldehyde (Fisher Chemical) in 0.1 M, PB (pH 7.2) for electron microscopic studies (3 animals). All procedures were approved by the University of Mississippi Medical Center or Washington National Primate Research Center Institutional Animal Care and Use committees and were performed in accordance with National Institutes of Health policies.

Livers were fixed for 5 hours in periodate-lysine-paraformaldehyde solution, which consisted of 0.2% sodium phosphate monobasic monohydrate (Fisher Scientific, Hampton, NH), 1.6% sodium phosphate dibasic heptahydrate (Fisher Scientific), 1% lysine (Sigma), 2% paraformaldehyde (Alfa Aesar, Haverhill, MA), and 0.22% sodium periodate (G-Biosciences, St. Louis, MO) at pH 7.4. Livers were then dehydrated in 30% sucrose (Sigma) overnight, embedded in OCT compound (Sakura, Torrance, CA), frozen via contact with dry ice, and cut into 10 µm sections using a Leica CM3050 S cryostat (Leica Biosystems, Buffalo Grove, IL). Liver sections were incubated in a solution consisting of 0.015% magnesium chloride (Sigma), 0.13% potassium ferricyanide (Ward’s Science, Rochester, NY), 0.17% potassium ferrocyanide (Chem Service, West Chester, PA), and 0.08% 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (Xgal; Teknova, Hollister, CA) overnight to stain for engrafted cells. Sections were then counterstained with hematoxylin (Ricca Chemical, Arlington, TX) and eosin (Electron Microscopy Sciences, Hatfield, PA) using standard procedures and mounted using Permount (Fisher Scientific). Bright-field photos were taken with a Zeiss Axiocam MRc 5 adapted to the photoport of a Zeiss Axiostar Plus microscope (Zeiss, Oberkochen, Germany).