Biotinylated donkey anti goat igg antibody

Draining LNs and spleens were fixed with 4% paraformaldehyde, washed with PBS, and then placed in Tissue-Tek OCT compound (Sakura) and snap-frozen in dry ice and ethanol. Cryosections 8 µm in thickness were affixed to MAS-GP–coated slides (Matsunami Glass) and stained at room temperature for 2 h or at 4°C for overnight with anti-IgD (11-26c.2a, FITC and PE), anti-CD4 (RM4-5, PE), anti-CD45.1 (A20, APC), anti-CD45.2 (104, APC), and rabbit anti–Bcl-6 (N-3; Santa Cruz Biotechnology, Inc.), which was detected by FITC-conjugated goat anti–rabbit IgG (SouthernBiotech) or by Alexa Fluor 488–conjugated goat anti–rabbit IgG (Invitrogen). Some samples were dehydrated with ethanol after sectioning instead of 4% paraformaldehyde fixation. Venus expression was detected by goat anti-GFP antibody (Rockland), biotinylated donkey anti–goat IgG antibody (Jackson ImmunoResearch Laboratories, Inc.), and TSA kit with HRP-streptavidin and Alexa Fluor 488 or Alexa Fluor 546 tyramide (Invitrogen). Images were acquired on BZ-9000 (Keyence), and processed with Photoshop (CS5.1; Adobe). ImageJ (National Institutes of Health) was used for the measurement of follicle and GC area for normalization of Tfh cell counts.