Anti madcam1

Lymph nodes were fixed for 3-4 h at 19 °C in freshly prepared 4% paraformaldehyde (Merck Millipore) under agitation. Organs mice were embedded in 4% low-melting agarose (Invitrogen) in PBS and serially sectioned with a vibratome (Leica VT-1200). 40-μm thick sections were blocked in PBS containing 10% FCS, 1 mg/ml anti-Fcγ receptor (BD Biosciences) and 0.1% Triton X-100 (Sigma). Tissues were incubated overnight at 4 °C with the following antibodies: anti-GFP (Aves), anti-DsRed (Living Colors), anti-B220, anti-IgD, anti-CD21/35, anti-PD1 (all from Thermo), anti-CD4, anti-Madcam1, anti-FcεR2a, anti-PDPN (BioLegend), anti-CCL21, anti-Lumican, anti-TRANCE, anti-CXCL13 (R&D Systems). Unconjugated or biotinylated antibodies were detected with the following secondary antibodies: Cy3-conjugated anti-rabbit-IgG, AlexaFluor647-conjugated anti-goat IgG, Dylight649-conjugated anti-syrian hamster-IgG, Dylight649-conjugated Streptavidin (all from Jackson Immunotools), or with AlexaFluor488-conjugated anti-chicken-IgG (Invitrogen). All antibodies used for histology are listed in the Life Sciences Reporting Summary. Microscopy was performed using a confocal microscope (LSM-710, Carl Zeiss), and images were recorded and processed with ZEN 2010 software (Carl Zeiss). Imaris Versions 8 and 9 were used for image analysis.