Bs 0296g af594

To assess the cell size of NMCMs, the NMCMs were fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.25% Triton X-100 for 10 min, followed by incubated with α-actinin primary antibody (Sigma Aldrich, A7811, 1:200, United States) overnight at 4°C. After washing, samples were incubated for 2 h at room temperature with fluorochrome-conjugated secondary antibodies (Bioss, bs-0296G-AF594, 1:400, China). Nuclei are stained with DAPI (Sigma Aldrich, D9542, 1:2000) for 5 min. Immunofluorescent micrographs were obtained using a laser-scanning confocal microscope (Leica Stellaris 5, Germany). The size of cardiomyocytes was analyzed by Image pro plus 6.0.

Mice were anesthetized and transcardially perfused with 0.9% saline and 4% formaldehyde, respectively. The brains were carefully removed, immediately dehydrated with 15% sucrose, and immersed in 30% sucrose the next day. Coronal brain sections of 10 μm thickness were incubated with 1% Triton X-100 for 30 min at room temperature. After blocking with 5% bovine serum albumin for 1 h at 37°C, sections were incubated overnight at 4°C with anti-GFAP mouse antibody (A00213, BOSTER, Inc.1:300), anti-NeuN mouse antibody (MAB377, Millipore, USA, 1:50) and anti-Iba-1 Chicken antibody (Cat234006, SYSY, Inc., Germany, 1:400). Sections were then incubated with Alexa Fluor@647-conjugated goat anti-chicken IgY (ab150171, Abcam, 1:400), Alexa Fluor 594-conjugated goat anti-mouse IgG (bs-0296G-AF594, Bioss, 1:300), or Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L; SA00006-1, Proteintech, 1:300) and incubated with DAPI (Sigma, USA, 1:200) to stain nuclei. All images were taken with an A1 + R laser confocal microscope (Nikon, Tokyo, Japan).