Comparison of Two Different Immunohistochemical Quadruple Staining Approaches to Identify Innate Lymphoid Cells in Formalin-fixed Paraffin-embedded Human Tissue

Lack of specific markers for innate lymphoid cells (ILCs) limit our knowledge on their spatial organization in situ. We compared two quadruple-color staining protocols for detection of the three principal human ILC subsets in formalin-fixed paraffin-embedded specimens. ILC subset–associated archetypical transcription factors (TFs) T-bet, GATA3, and RORγt were used as positive identifiers in combination with lymphoid lineage markers to exclude non-ILCs. One method (“virtual quadruple staining”) comprised of iterative single stainings on the same section performing digital scanning and subsequent immunoglobulin and chromogen stripping after each staining round. The second technique (“true-color quadruple staining”) comprised sequential double stainings with permanent colors. Both protocols appeared suitable for accurate detection of each ILC subset, and as added result, concomitant visualization of their T cell subset counterpart. Only true-color quadruple staining enabled simultaneous detection of all three ILC subsets within one section. Furthermore, we found that type 3 and type 1 ILCs (ILC1s) represent the major subsets in colon and that part of the ILC1s typically colocalizes with blood vessels. Our data highlight the utility of TFs combined with lineage markers for the identification of ILC subsets and proposed workflow opens the way to gain deeper insight of their anatomical distribution.