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

Area of Science:

• Immunology
• Cell Biology
• Histology

Background:

• The in vitro lymphocyte binding assay (HEV assay) is crucial for studying lymphocyte homing to high endothelial venules (HEVs).
• Current methods using fluorescence-labeled lymphocytes require fluorescence microscopy, preventing counterstaining and easy identification of HEVs lacking lymphocytes.
• Existing HEV preparations are not stable for storage, necessitating rapid evaluation.

Purpose of the Study:

• To develop an improved technique for evaluating HEV preparations in lymphocyte binding assays.
• To enable light microscopy evaluation of counterstained HEV sections with fluorescence-labeled lymphocytes.
• To allow for simultaneous determination of lymphocyte phenotypes and permanent storage of preparations.

Main Methods:

• Development of a novel technique for preparing HEV sections compatible with light microscopy.
• Utilizing fluorescence-labeled standard lymphocytes for assay standardization.
• Implementing counterstaining for enhanced visualization and surface antigen staining for phenotype analysis.

Main Results:

• The improved technique allows HEV preparations with fluorescence-labeled lymphocytes to be evaluated using light microscopy.
• Counterstaining facilitates easier recognition of HEVs and adherent lymphocytes.
• Simultaneous staining for surface antigens enables determination of lymphocyte phenotypes.
• The resulting preparations are permanent, allowing for later analysis and storage.

Conclusions:

• The improved HEV assay technique enhances the evaluation of in vitro lymphocyte binding.
• This method permits detailed analysis of lymphocyte-HEV interactions, including phenotype determination.
• The ability to store preparations permanently offers greater flexibility and reproducibility in research.