Fitc labeled streptavidin

The detailed methods for fluorescence immunohistochemistry employed in this study have been described previously [22 (link)]. Frozen sections were cut to 6 μm thickness and fixed in cold acetone and 10% buffered formalin. A polyclonal rabbit antibody against Cx32 (Thermo Fischer Scientific Inc., Waltham, MA) was used with biotin-conjugated anti-rabbit IgG and TRITC-labeled streptavidin (Thermo Fischer Scientific Inc.) to visualize the endogenous proteins using an image analyzer (Keyence). A monoclonal mouse antibody against Cx26 (Thermo Fischer Scientific Inc.) was used with biotin-conjugated anti-mouse IgG and FITC-labeled streptavidin (Thermo Fischer Scientific Inc.).

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.