Madcam 1 meca 367

Organs were embedded in Tissue-Tek O.C.T Compound (Electron Microscopy Science) and frozen in liquid nitrogen. Six to 8 μm sections were cut, fixed in cold acetone and then blocked with 2% BSA. The following antibodies were used: mCLCA1 (hamster mAb 10.1.1), Lyve-1-A488 (ALY7, eBioscience), ITGA2b-APC (MWReg30, Biolegend), Prox-1 (polyclonal goat, R&D Systems), GPIbß (RAM1-A645), MAdCAM-1 (MECA-367, BD-Pharmingen), fibronectin (Rabbit polyclonal, Patricia Simon-Assmann, UMR-S 1109 INSERM, Strasbourg), RANKL (IK22.5, Rat IgG2a, [22 (link)]), goat anti-rabbit-A488 (Molecular Probes), goat anti-hamster-A488/A546 (Molecular Probes), donkey anti-rat-Cy3 (Jackson) or streptavidin A546 or A647 (Molecular Probes). Sections were mounted using DAKO mounting medium (Dako, Hamburg, Germany). Images were acquired and treated as noted above.

ICAM-1 (Cat. No. 09351D, 1:500) was purchased from Pharmingen, San Diego, CA, USA; VCAM-1 (Cat. No. 553329, 1:500) was purchased from BD Biosciences, San Diego, CA, USA; MAdCAM-1 (MECA-367, Cat. No. 09721D, 1:500) was purchased from BD Pharmingen, San Diego, CA, USA; PECAM-1 (Cat. No. 550274, 1:500) was purchased from BD Pharmingen; E-selectin (Cat. No. 553748, 1:500) was purchased from BD Biosciences; P-selectin (Cat. No. 09480D, 1:1000) was purchased from Pharmingen.