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ILCs interact with multiple cell types within their local environment to integrate a wealth of different signals into coordinated responses that regulate tissue homeostasis as well as immune responses upon challenge. While the development and function of ILCs has been extensively studied, principally using flow cytometry, there is limited understanding of the precise composition of cellular niches within which ILCs reside. While this might be optimally studied using dynamic live imaging approaches, immunofluorescence staining of tissue sections can provide fundamental basic information regarding the nature of these microenvironments. Here, a methodology enabling the identification of murine and human ILC populations in frozen tissue sections using immunofluorescence is described.

Innate lymphoid cells (ILCs) are present in most tissues within the body but have been most extensively studied within mucosal barriers such as the lung and intestine. Isolation of immune cells from such tissues requires enzymatic digestion, and the number and composition of the cells released are dependent upon robust protocols tailored to the tissue of study. Here, detailed methodologies to isolate ILCs from various barrier sites and their draining lymph nodes (LNs) are described. Flow cytometry staining and gating strategies for identification and quantification of ILCs are then provided. Combined, these provide an efficient means to study ILCs within the small intestine lamina propria, lung, ear skin, and LNs.