Perfusion Fixation for Cryosectioning

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.