Journal: bioRxiv

Article Title: Quantitative Multicolored Deep Imaging of Human Bones Reveals a Composite Osteo-Sinusoidal Niche for Mesenchymal Stromal Cells

doi: 10.1101/2025.10.07.680053

Figure Lengend Snippet: a-g, Three-dimensional in situ hybridization of CXCL12 mRNA (red) combined with immunostaining for CD31-positive vasculature (yellow) and perilipin-positive adipocytes (white) in young ( a-d ) and aged ( e-g ) human bone. The depth of the scans was 3 mm for young and 3.5 mm for aged bone (z-axis). Bone (cyan) was visualized on the basis of autofluorescence (omitted in a , c , and f to simplify visualization). b,c, Three-dimensional projections at a higher level of magnification, and d two-dimensional optical section from a . f , Three-dimensional projections at higher magnifications along with a two-dimensional optical section ( g ) from e . The purple arrowheads in d and g point to CXCL12+ cells on bone or adipocyte surfaces. h-k , Cumulative distribution functions (CDF, solid lines) of CXCL12+ cells relative to the adipocyte surface ( h , i, n=3 for young patients, n=6 for aged patients) and bone surface ( j , k, n=6 for young patients, n=6 for aged patients) as compared with random distributions of these same spots within the same scans (dashed lines). The shadow areas outline 95% confidence intervals for the mean (n=patients), and inserts depict the CDF at a 50-μm distance with data color-coded by individual patients. Real–measured data, Rand–randomized distribution. The randomized distribution of spots was performed using Imaris software. The comparison of CD functions was performed using the Kolmogorov-Smirnov test, whereas the CDF values at a 50-μm distance were compared using a paired t-test. A small number of patients in h,i is caused by the limited tissue availability and priorities toward other analyses.

Article Snippet: Pretreated samples were prehybridized with hybridization buffer (30% formamide, 5x sodium chloride sodium citrate (SSC), 9 mM citric acid (pH6.0), 0.1% Tween 20, 50 μg/mL heparin, 1x Denhardt’s solution, 10% Dextran sulfate, from Molecular Instruments) for 2 hours on a 37°C shaker at 200 rpm (rotations per minute).

Techniques: In Situ Hybridization, Immunostaining, Software, Comparison