Immunocytochemistry was performed as previously described [20 (link)]. Briefly, the cells were fixed, permeabilized, blocked, and incubated with primary antibodies against SARS-CoV-2 nucleocapsid (#GTX135357, 1:100; GeneTex, Irvine, CA, USA), SARS spike glycoprotein (1A9, #ab273433, 1:100; Abcam, Cambridge, UK), CD31 (#ab28364, 1:25; Abcam) [29 (link)], CLDN5 (4C3C2, #35-2500, 1:25; Thermo Fisher Scientific) [29 (link)], cleaved caspase-3 (5A1E, #9664S, 1:500; Cell Signaling Technology), phospho-GYS1 (1D1, # CSB-RA010078A641PHHU, 1:100; Cusabio, Wuhan, China), and β-catenin (15B8, #37447S, 1:3000; Cell Signaling Technology, Danvers, MA, USA) at 4 °C. The cells were then incubated with Alexa 488-conjugated (1:200; Thermo Fisher Scientific) or Alexa 594-conjugated (1:200; Thermo Fisher Scientific) secondary antibodies for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole DAPI (Nacalai Tesque Inc.). The cells were mounted in SlowFade (Thermo Fisher Scientific) and examined under a confocal laser-scanning microscope (Nikon A1; Nikon, Tokyo, Japan).
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