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,b , Colocalization of LEPR protein (antibody staining, pseudocoloured yellow) and CXCL12 mRNA (HCR RNA-FISH, red) in representative 2D optical sections from ( a ) a 3D light-sheet scan and ( b ) a confocal scan of tissue-cleared human bone. Left panels show LEPR omitted; right panels include all channels. Nuclei (YOPRO, green) and bone matrix (autofluorescence at 405 nm, cyan) were visualized in ( a ) but not ( b ) due to sample bleaching. c , Confocal image of a 6-μm thick section from decalcified human bone stained for LEPR (green), CXCL12 mRNA (red), and counterstained with DAPI (blue). Single and merged channels are shown. d–f , Nuclear staining of aged human bone with YOPRO1 (green). d, Raw YOPRO+ signals in a light-sheet scan. e, YOPRO+ nuclei overlaid with cell positions detected by the Imaris Spot function. f, Optical sections showing overlay of YOPRO+ nuclei with segmented cell masks (grey circles). g–l , Detection of nucleated CXCL12+ cells in aged bone. g–i, Representative 2D optical sections from a 3D light-sheet scan showing CXCL12 mRNA (HCR, red), CD31+ vessels (yellow), and YOPRO1+ nuclei (green). CXCL12 signal is omitted in g and present in h ; i , colocalized CXCL12+YOPRO+ cells detected by Imaris Spot function are marked by grey circles. j–l , 3D projections of the same region as a raw scan ( j ) and detected double-positive cells at low ( k ) and high ( l ) magnification, with double-positive cells represented as green spheres. j , CD31 and YOPRO channels only; k,l , all channels including CXCL12. All images are cropped from large tissue volumes (∼2 × 2 × 2 mm) scanned by light-sheet microscopy.
Article Snippet: Following HCR, tissue sections were blocked using the same staining buffer as used in DeepBone, then incubated with primary antibodies against LEPR (conjugated to AlexaFluor488; #MAB867, R&D Systems) or Ki67 (conjugated to AlexaFluor546; SolA15, Thermofisher).
Techniques: Staining, Microscopy