Anti tnp g235 1

Splenocytes were made into a single‐cell suspension, red cell lysed and processed on ice during staining. The following antibodies were purchased from BioLegend (San Diego, CA): anti‐CD1d (1B1), anti‐CD3e (145‐2C11), anti‐CD21/35 (7E9), anti‐CD23 (B3B4), anti‐CD45R/B220 (RA3‐6B2), anti‐CD93 (AA4.1), anti‐CD185/CXCR5 (L138D7). The following antibodies were purchased from BD Biosciences (Oxford, UK): anti‐CD16/32 (2.4G2) and anti‐TNP (G235‐1). Anti‐S1P₁/EDG‐1 (713412) was purchased from R&D Systems (Minneapolis, MN). After immunostaining, cells were analysed using an LSR Fortessa with diva software (BD Biosciences, London, UK). Cells were gated on lymphocytes and doublets were removed, then data were analysed using flowjo (FlowJo, LLC, Ashland, OR).

Frozen sections 6–8 μm thick were fixed in ice‐cold acetone, rehydrated in PBS and blocked with normal horse serum before antibody application. The following antibodies were purchased from BioLegend: anti‐CD1d (1B1), anti‐CD4 (RM4‐5), anti‐CD21/35 (7E9) and anti‐CD45R/B220 (RA3‐6B2). The following antibodies were purchased from BD Biosciences: anti‐CD35 (8C12), anti‐MAdCAM‐1 (MECA‐367) and anti‐TNP (G235‐1). Anti‐CD169 (MOMA‐1) and anti‐MARCO (ED31) were purchased from Bio‐Rad (Hemel Hempstead, UK). Anti‐CD209b/SIGNR1 (eBio22D1) and phycoerythrin (PE)‐conjugated anti‐Armenian hamster IgG were purchased from eBiosciences (ThermoFischer, Loughborough, UK). Anti‐CXCL13 (polyclonal) was purchased from R&D Systems. Streptavidin Alexa Fluor 594, goat anti‐rat IgG (H+L) Alexa Fluor 594, donkey anti‐goat IgG (H+L) Alexa Fluor 647 and goat anti‐rat IgG (H+L) Alexa Fluor 488 were purchased from ThermoFisher Scientific (Waltham, MA). Dako fluorescent mounting medium (Agilent, Santa Clara, CA) was used to apply coverslips before image acquisition. A Zeiss LSM5 Pascal (Carl Zeiss, Oberkochen, Germany) upright microscope with zen software (Rochdale, UK) was used for image collection.