Anti ccl21

In the mouse experiment, sections were incubated with the primary antibody anti-type II collagen (Daiichi Fine Chemical). In the rabbit experiment, sections were incubated with the primary antibodies anti-type I (abcam) and anti-type II collagen (Fuji Pharm.). The samples were incubated with a biotinylated secondary antibody and visualized by the precipitation of diaminobenzidine (DAKO). For CCL21 and CCR7 evaluation, sections were incubated with the primary antibodies anti-CCL21 (R&D), anti-CCR7 (Abcam), and staining was visualized with Alexa Fluor 568 (Invitrogen), FITC Conjugated AffiniPure (Jackson Immuno Research). Nuclei were stained with DAPI (Invitrogen).

FITC-, PE-, PerCP-Cy5.5–, Alexa Fluor 647–, APC-, eFluor 450–, and eFluor 660–conjugated antibodies specific for AnnexinV, B220 (RA3-6B2), CD3 (145-2C11), CD4 (GK1.5, RM4-5), CD8 (53-6.7), CD31 (390), CD44 (IM7), CD54 (YN1/1.7.4), CD62L (Mel-14), CD69 (H1.2F3), CD106 (429), LYVE1 (ALY7), and PNAd (MECA-79) were purchased from BioLegend or Invitrogen. Anti-CCL21 and PE-labeled anti-S1P1 (713412) were purchased from R&D Systems. FITC-labelled TCRβ (H57-597) was purchased from BD Biosciences. Alexa Fluor 488– or 633–conjugated goat anti-mouse, anti-rat, or anti-human IgG (1–2 μg/mL) was purchased from Jackson ImmunoResearch Laboratories. Anti-LFA1 (KBA) and anti-CD62L (MEL) for neutralization was prepared from our laboratory. CFSE (5- or 6-(N-succinimidyloxycarbonyl)-fluorescein 3′,6′-diacetate), CMTMR ((5-(and 6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine), and CMF2HC (4-chloromethyl-6,8-difluoro-7-hydroxycoumarin) dye for cell labeling were obtained from Invitrogen. Recombinant CCL21 was purchased from R&D Systems and S1P and FTY720 were obtained from Cayman Chemicals.

To induce intestinal inflammation, mice were injected intravenously with purified flagellin from Salmonella typhimurium (FLA-ST Ultrapure, 5 μg; Invivogen) 5 h before analysis. For microbiota depletion, mice were orally treated for 2 weeks with a combination of antibiotics, administered in their drinking water, including ampicillin (1 mg ml⁻¹), streptomycin (5 mg ml⁻¹), colistin (1 mg ml⁻¹) and sucrose (25 mg l⁻¹), all from Sigma-Aldrich. For chemokine neutralization, mice were injected intravenously during imaging with a mixture of Hoechst 33342 (see above) to control intravenous injection and the following monoclonal blocking antibodies (50 µg): anti-CXCL12 (catalog no. MAB310); anti-CXCL16 (catalog no. MAB503); anti-CCL21 (catalog no. MAB4572) and anti-CCL25 (catalog no. MAB481), all from R&D Systems. Control mice were injected intravenously in accordance with the following isotype controls (50 µg): mouse IgG1 (catalog no. MAB002); rat IgG2a (catalog no. MAB006); and rat IgG2b (catalog no. MAB0061), all from R&D Systems.