Multicolor simultaneous full-bone quantitative imaging of HSCs and multiple niche populations. (A) Experimental flow and duration of large-volume, multicolor, quantitative confocal imaging. See description in “Analysis” in “Methods.” (B) Six-color immunostaining of 220- to 250-μm–thick, full-bone, longitudinal sections of mouse femur (top) and sternum (bottom) from an α-catulin^GFP/+ reporter mouse. Sections stained for collagen.1/osteopontin-CF405S (trabecular and cortical bone), GFAP fab-Alexa-594 (nonmyelinated Schwann cells), laminin-CF633 (vasculature), and GP1bβ-CF680 (MKs). HSC^α-cats were marked by GFP (amplified by biotin-streptavidin 488) and cKit-CF555 expression. CF, cyanine-based fluorescent dyes, SA, streptavidin. (C) Overview of niche components, respective marker combinations, and staining schemes. The number of sections imaged for each niche population, marker, bone type, and HSC reporter system are shown. (D-G) High-magnification images of multicolor staining schemes for niche components. (D) Femoral area (enlargement of red dashed square appearing in panel B) showing nonmyelinated Schwann cells (Sc, yellow, GFAP); cortical bone (white, Col.1/Opn); MKs (magenta, GP1bβ); and different vessel types marked by laminin: central sinus, sinusoids (S), arteries (A), arterioles (a) and transition zone vessels (TZ). (E) Trabecular and cortical bone (white, Col.1/Opn), adipocytes (yellow; boron dipyrromethene, BODIPY TRx), and BM vasculature (cyan, laminin). (F) Cortical bone (white, Col.1/Opn), sinusoids (cyan, CD105), and Cxcl12⁺ stroma (yellow). (G) Trabecular bone (white, Col.1/Opn), Sca1⁺ arteries (cyan), and NG2⁺ periarteriolar cells (yellow) after computationally masking all NG2⁺ objects outside Sca1⁺ arteriolar signal to appear black. Scale bars: 1000 μm (B), 100 μm (D), 50 μm (E, G), and 30 μm (F).