Objective: To develop a method for immunohistochemical staining of three different T lymphocyte antigens (CD4 or CD8, CD57 and TCRβ) on the same tissue section and to determine whether tissues have been infiltrated with T lymphocytes expressing these markers. Study Design: Commercially available antibodies were tested for immunohistochemical usefulness in a dye-based conventional single-immunostaining method after antigen retrieval on paraffin-embedded human lymph nodes. We searched for the combination of antibodies that could detect T lymphocyte antigens on the same section without any cross-reactivity and that have fluorescent signals robust enough to overcome paraffin autofluorescence. Results: Application of the antigen retrieval technique and the Sudan black B quenching technique enabled staining of paraffin-embedded tissue sections with fluorescent-labeled secondary antibodies. The combination of primary and secondary antibodies that could simultaneously detect the T lymphocyte antigens CD4 or CD8, CD57 and TCRβ in histochemical analysis of a paraffin-embedded human lymph node section was established, and was successfully applied to a human tissue section infiltrated with T lymphocytes that express these markers. Conclusion: The antibodies listed here would be helpful for histopathologists who wish to investigate T lymphocytes in the paraffin-embedded sections that have accumulated in pathology labs throughout the world.