Endomucin

After the last time point of in vivo U‐LSOM imaging, mice were euthanized and their entire skull bone extracted and sliced as described.⁽²⁷⁾ Consecutive slices of 150 μm thickness were generated throughout the entire bone. For confocal imaging, slices covering or proximal to the coronal suture were selected. For immunostaining, individual slices were first incubated in blocking solution (0.2% Triton X‐100, 10% donkey serum in PBS) overnight at 4°C and then stained with primary antibodies (Endomucin, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA; Sc‐65495) in blocking solution for 3 days at 4°C. Tissues were then washed in 0.2% Triton‐X‐100/PBS and stained with secondary antibodies (Alexa Fluor 488 donkey anti‐rat immunoglobulin G [IgG] Antibody, 1:400; Thermo Fisher Scientific, Waltham, MA, USA; A‐21208) for another 3 days at 4°C in blocking solution. Immunostained slices were washed in 0.2% Triton‐X‐100/PBS overnight and incubated in RapiClear 1.52 for 12 to 16 hours. Confocal microscopy was performed with a 10× objective (HCX PL FLUOTAR; Leica, Wetzlar, Germany) on a Leica SP8 Leica confocal microscope. Image stacks (x: 3070–3080 μm, y: 9300–10300 μm, z: 125–145 μm) were acquired with a pixel size of 2.27 μm and a Z‐step size of 3 μm. Imaris software (Oxford Instruments plc, Abingdon, UK) was used to render confocal image stacks into 3D reconstructions (Fig. 3B).