Characterization of Cd34 ^creER ;R26 ^tdTomato mice. (A) Schematic of the design of Cd34^creER mice. (B) Three-dimensional reconstruction of whole liver from Cd34^creER;R26^tdTomato mice. Samples were cleared using the PEGASOS method, and images were captured using a light-sheet microscope. Scale bar = 500 µm. (C) Quantification of the percentage of CD34⁺ fibroblasts that were Tomato⁺ on the liver sections from Cd34^creER;R26^tdTomato mice. (D) Confocal image showing Tomato expression in the portal space of Cd34^creER;R26^tdTomato mice. Anti-Thy-1 staining identified portal fibroblasts surrounding portal veins and bile ducts, while anti-aSma staining labeled VSMCs surrounding arteries and portal veins (upper). Bile ducts and arteries were distinguished by anti-Epcam and anti-aSma antibodies (bottom). Arrows depict the portal vein; arrowheads depict the hepatic artery. Scale bar = 50 µm. (E–G) On-slide staining of non-parenchymal liver cells from Cd34^creER;R26^tdTomato mice with anti-Col1 (E), anti-Myh11 (F), and anti-Reln (G) antibodies, respectively. Cells were isolated by gradient centrifugation and were mounted on poly-D-lysine–coated slides. Scale bar = 25 µm. (H) Confocal image showing no Tomato expression in Hnf4a⁺ hepatocytes in Cd34^creER;R26^tdTomato mice. Scale bar = 50 µm. (I) Flow cytometric analysis of cells from whole bone marrow showing that ∼25% of all hematopoietic stem cells were Tomato⁺ in Cd34^creER;R26^tdTomato mice.