A, AB, B, and DN cells predominantly line trabecular bone. Shown are representative images of multiplexed IHC in Prrx1:EYFP mice tibias. Color vision deficiency compatible images are shown in supplemental Figure 8. Mouse tibial sections were stained for the same markers used to identify the distinct microenvironment cell populations by FACS, scanned using a Vectra Automated Quantitative Pathology Imaging System, and visualized using Fiji software. Shown are Sca-1 (Opal 520, red), EYFP (Opal 540, green), CD51 (Opal 650, yellow), PDGFR⍺ (Opal 570, cyan), PDGFRβ (Opal 620, magenta), nuclei (4′,6-diamidino-2-phenylindole [DAPI], white); hematopoietic lineage staining was excluded for visualization purposes but is shown in supplemental Figure 2. All images are shown as a composite image with all markers and individual staining for each field of view shown on the right. Three regions of interest were examined: growth plate (A-B), trabecular bone (C), and metaphyseal cortical bone (D). Dotted red lines indicate the border of trabecular and cortical bone. Colored arrowheads indicate populations A (red arrowhead; PDGFR⍺⁺PDGFRβ^–), AB (purple arrowhead; PDGFR⍺⁺PDGFRβ⁺), B (blue arrowhead; PDGFR⍺^–β⁺) and DN (orange arrowhead; PDGFR⍺^–PDGFRβ^–), which are all Sca-1^–EYFP⁺CD51⁺lin^–. (E) Representative single-cell images of A, AB, B, and DN microenvironment cells. Arrowheads point to the single cell shown on each individual panel. Scale bar, 100 µm. Images have been thresholded, brightness and contrast were adjusted equally for visualization purposes, and all analyses were performed on raw data. C, cortical bone; GP,  growth plate; T, trabecular bone.