Anti lyve 1

Frozen sections were prepared from the tail at 28 days after the ablation surgery. The 8‐μm‐thick sections were fixed in 4% paraformaldehyde, washed twice with PBS, and blocked with 0.5% FBS albumin at room temperature for 1 hour. Sections were then incubated with primary antibodies, anti‐LYVE‐1 (lymphatic vessel endothelial hyaluronan receptor 1) (1:125; Acris) or phospho‐Akt (or Protein kinase B) (Cell Signaling Technology, CST; number 4060, 1:400), and anti‐mouse podoplanin (1:250; R&D Systems) antibodies, at 4 °C overnight, followed by incubation with secondary antibodies, Alexa‐Fluor 488‐conjugated anti‐rabbit (1:1000; Thermo Fisher Scientific) and Alexa‐Fluor 594‐conjugated anti‐goat (1:1000; Thermo Fisher Scientific) antibodies, respectively, at room temperature for 1 hour.¹⁹, ²⁰, ²¹ The capillary density of lymphatic vessels was evaluated as LYVE‐1 and podoplanin double‐positive cells per field by previously established methods.¹⁷, ¹⁹, ²¹ The nuclei were stained using 4′,6‐diamidino‐2‐phenylindole (1:1000; Dojindo), and macrophages were detected by phycoerythrin‐labeled anti‐mouse F4/80 monoclonal antibody (1:1000; BioLegend). Images were visualized using a BZ‐X710 fluorescent microscope (KEYENCE), with a ×20 objective lens. Positive cells per field were counted.