The in vitro lymphocyte/endothelium binding assay. An improved method employing light microscopy.

The in vitro lymphocyte binding assay (HEV assay) has proved to be a useful approach for examining the first step of lymphocyte migration, i.e., homing to organs containing high endothelial venules (HEVs). Since fluorescence-labelled standard lymphocytes are usually included in each assay to account for day-to-day variations, HEV preparations have to be evaluated by fluorescence microscopy. Thus no counterstaining can be performed and HEVs without adherent lymphocytes cannot easily be recognized. Because the preparations are not suitable for storage they must be evaluated within a short time. In this study an improved technique is described which permits HEV preparations made with fluorescence-labelled standard lymphocytes to be evaluated by light microscopy in counterstained sections. The phenotypes of the sample lymphocytes can be determined by staining for surface antigens on the same slides and the preparations obtained are permanent.