Recent technical advances, such as single-cell RNA sequencing and mass cytometry, improve identification of cell types and subsets in a range of healthy and diseased tissues at the expense of their cellular and molecular context. Here, we present a protocol for in situ multispectral imaging to map myeloid cell heterogeneity in tissue cryosections, describing steps for cutting sequential sections, antibody titration, and building a spectral library. We then detail procedures for …