Mouse anti human igd bv421

Unfixed pediatric tonsil samples frozen in OCT medium were cut into 9μm thick sections using a cryostat (Leica) and stored at −80°C. Tonsil sections were brought to room temperature and then fixed at −20°C for 10 min in pre-cooled 50:50 Acetone:Methanol solution. Sections were dried and then rehydrated in PBS for 5 min with gentle shaking. The sections were then blocked in 2% normal mouse serum (Invitrogen) for 1 hour and subsequently stained overnight at 4°C with mouse anti-human TCR Vα7.2-PE (Clone 3C10, Biolegend), mouse anti-human CD161-AF488 (Clone NKR-P1A, Biolegend), mouse anti-human IgD-BV421 (Clone IA6–2, Biolegend) and mouse anti-human PD-1-APC (Clone EH1202H7, Biolegend) all diluted 1:100. Sections were washed 3x in PBS and mounted using ProLong Gold Antifade Mounting media (Thermo Fisher). Full tonsil cross section images were acquired using the Leica Sp8 Confocal microscope and then processed and analyzed using ImageJ.