Lymphocyte binding to frozen LN sections. Wild-type lymphocytes were allowed to bind to frozen sections of MLNs of littermate control (+/+) and Slc35c1^−/− (−/−) mice under mild rotation. Following fixation, HEVs were stained with anti–MAdCAM-1 mAb MECA 367. Isotype control mAb incubation gave no staining. (A) Micrograph of typical sections following lymphocyte binding and HEV staining. (B) Quantification of lymphocyte binding to HEVs. Binding is expressed as the number of HEV-bound cells per square millimeter of HEV area on the sections. Only cells bound to stained areas were considered. Background binding (cells per square millimeter of parenchyma) was subtracted. (Inset) L-selectin dependence of lymphocyte binding to sections of Slc35c1-deficient MLNs. Lymphocytes were preincubated with a nonblocking isotype control antibody directed against CD99 (“Methods”) or with the L-selectin–specific mAb Mel-14 before they were allowed to bind to the sections. For each type of mouse the data are from 4 animals and 7 MLNs with a total of 90 HEVs.