Immunohistochemical Staining of Cryosections

Cryosections of 7 μm were fixed and stained immunohistochemically as described^{17 (link),31 (link)} with: FITC-conjugated anti-IgD (clone 11-26c.2a, BD Biosciences), biotin-conjugated anti-CD45.1 (clone A20, Biolegend, San Diego, CA, USA), biotin-conjugated anti-DCIR2 (clone 33D1, Biolegend) or biotin-conjugated anti-CD25 (clone PC61.5, BD Biosciences) followed by HRP-conjugated anti-FITC, AP-conjugated anti-FITC, and/or AP-conjugated SA (Jackson Immunoresearch, Newmarket, Suffolk, UK). For staining of DCIR2 and CD25, a tyramide amplification kit was used (TSA Biotin System; Perkin Elmer; Waltham, MA, USA). For immunofluorescence, staining was performed with biotin conjugated anti-CD45.1, rabbit anti-GFP (Molecular Probes, Eugene, OR, USA), and goat anti-mouse IgD (GAM/IGD(FC)/7S, Cedarlane Laboratories), followed by AMCA-conjugated donkey anti-goat IgG (Jackson Immunoresearch), Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 647-conjugated streptavidin (Invitrogen). Images were captured with a Zeiss AxioOberver Z1 inverted microscope.