| Characterizing the cellular participants in tissue immune responses is critical for understanding infection, cancer, autoimmunity, allergy, graft rejection, and other immunological processes. Leukocytes recirculate through blood vessels before localizing to tissue sites of immune responses. Thus, in experimental animal models, tissues are often perfused to putatively remove blood-borne leukocytes that may conflate analysis. Here, we develop and validate an intravascular staining methodology that distinguishes between vascular and tissue-localized cells of the immune system. We demonstrate that perfusion both fails to remove many blood-borne leukocytes and also may remove tissue-localized populations of interest, and we provide examples of how this issue distorts interpretation of leukocyte differentiation state, migration, and phenotype in healthy mice, as well as those responding to viral or Mycobacterium tuberculosis infection or tumor challenge. Additionally, we utilize intravascular staining to examine resident memory T cells in non-lymphoid tissues, such as the lung, liver and female reproductive tract. This study highlights the breadth and gravity of the issues regarding tissue leukocyte composition, outlines simple methods for identification of various intravascular leukocyte populations, reviews the limitations of the technology, and demonstrates that these methods should be routinely adopted in lieu of perfusion for interpretable and accurate analyses of immune responses in many tissues. |
