Thymi were fixed with 4% paraformaldehyde overnight, transferred to 30% sucrose solution for 24 h, and snap frozen in optimum cutting temperature compound. Sections (7 μm) were blocked with 5% bovine serum albumin and Fc block (anti-CD16/CD32; 2.4G2, Tonbo Biosciences) in dilution 1 : 100 for 1 h at room temperature (RT) prior to staining. The sections were incubated with Rabbit-anti-b5t in dilution 1 : 200 (MBL International) and fluorescein-labeled Ulex europaeus agglutinin I (UEA-I) (Vector Laboratories) at 4 °C overnight, followed by Goat-anti-Rabbit-AF555 in dilution 1 : 500 for 1 h at RT (Thermo Fisher Scientific). Sections were next stained with 4′,6-diamidino-2-phenylindole and mounted using ProLong antifade mounting medium (Life Technologies). Images were acquired using a Leica DM6000B epifluorescent microscope.

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