Bv510 labeled anti cd4 rm4 5

Tissue specimens submerged in OCT compound were quickly frozen in liquid nitrogen and cut into 10 μm thickness. Frozen tissue sections were then fixed in cold acetone for 30 min at −20 °C, blocked with 1% BSA and Fc-blocker (2.4G2; 1:100; BD Biosciences) in PBS. The tissue sections were then stained with biotinylated PNA (Cat. no. BA-0074; 1:20; Vector laboratories), followed by staining with BV510-labeled anti-CD4 (RM4-5; 1:100; BD Biosciences), APC-labeled anti-IgD (11-26c; 1:100; Thermo Fisher Scientific), and AF488-conjugated streptavidin (1:500; Invitrogen). Then the slides were washed at least three times with PBS. Coverslips were mounted on slides using an antifade kit (Beyotime Biotechnology) and then examined using an Andor Dragonfly confocal microscope. The images were processed with Imaris (v8.1; Bitplane) and Image J (v1.52 g; NIH).

Spleens from mice infected with LCMV Armstrong were snap-frozen in OCT medium (Sakura Finetek) by liquid nitrogen. Ten-micrometer-thick sections were cut using a Cryostat Microtome System. Tissue sections were fixed with cold acetone for 30 min at −20°C, blocked with 5% BSA, and stained with biotin-PNA (Vector Laboratories), APC-labeled anti-IgD (11–26 c.2a; Thermo Fisher Scientific), and BV510-labeled anti-CD4 (RM4-5; BD Biosciences), followed by FITC-labeled streptavidin (Thermo Fisher Scientific). After each step, the slides were washed at least three times with PBS. Sections were fixed and mounted with an antifade kit (P0123, Beyotime Biotechnology) and then examined using a confocal fluorescence microscope. The images were processed with Imaris and Image J software.