Richard allan scientific neg 50 frozen section medium

After X-ray imaging, the calvaria were placed in a 30% sucrose solution in PBS (pH 7.4) for 1 day. The tissues were then positioned in Richard-Allan Scientific™ Neg-50™ frozen section medium (Thermo Scientific). Cryosections (5 μm) through the non-decalcified calvaria were obtained on a Leica CM3050-S cryostat (Leica, Wetzlar) using a disposable steel blade (Thermo Scientific) and tape transfer process (cryofilm type IIC (10), Section-Lab Co. LTD) as previously described. The slides were then prepared with 50% glycerin in PBS as the mounting medium. The sections were initially imaged for differential interference contrast (DIC) using the Zeiss Axio Scan.Z1 (Carl Zeiss Microscopy). Next, the endogenous fluorescence of the Col3.6Topaz and Col3.6Cyan fluorescent reporters, and the AC mineralization label were imaged. The sections were then sequentially stained and imaged for TRAP enzymatic activity, ALP, DAPI, and toluidine blue O, as previously reported^{24 (link),78 (link),90} . This sequence was possible because the cryofilm tape adheres to the tissue and allows for the coverslip to be removed between the imaging steps without damaging the section.

Retinal explants were washed in PBS (2.7 mM KCl, 140 mM NaCl, 10 mM phosphate, pH 7.4) once. Subsequently, they were submerged in 4% (w/v) paraformaldehyde and incubated for 1 hr at room temperature. Retinae were washed in PBS twice before they were put in 30% (w/v) sucrose overnight. Single retinae were then embedded in Richard‐Allan Scientific^™ Neg‐50^™ Frozen Section Medium (Thermo Fisher Scientific, Waltham, MA, USA) and fast frozen in liquid nitrogen. About 10 μm cryosections were cut. Immunolabelling with anti‐ATP1B2 and antiretinoschisin antibodies was performed as described by 23. Cone visualization was performed with Alexa 488‐conjugated peanut agglutinin (1:250, PNA; Invitrogen). Rhodopsin staining was performed with Rho‐1D4 antibody (1:1000), kindly provided by Prof. Robert Molday, University of British Columbia, Vancouver, Canada. The sections were counterstained with 4′,6‐diamidino‐2‐phenylindol (DAPI, 1:1000; Molecular Probes, Leiden, the Netherlands). Images were taken with custom‐made VisiScope CSU‐X1 Confocal System (Visitron Systems, Puchheim, Germany) equipped with high‐resolution sCMOS camera (PCO AG, Kehlheim, Germany).