Deep imaging of LepR^(+)stromal cells in optically cleared murine bone hemisections  

作　　者：Yuehan Ni Jiamiao Wu Fengqi Liu Yating Yi Xiangjiao Meng Xiang Gao Luyi Xiao Weiwei Zhou Zexi Chen Peng Chu Dan Xing Ye Yuan Donghui Ding Ge Shen Min Yang Ronjie Wu Ling Wang Luiza Martins Nascentes Melo Sien Lin Xiaoguang Cheng Gang Li Alpaslan Tasdogan Jessalyn M.Ubellacker Hu Zhao Shentong Fang Bo Shen 

机构地区：[1]College of Life Sciences,Beijing Normal University,100875,Beijing,China [2]National Institute of Biological Sciences,Beijing(NIBS),102206,Beijing,China [3]Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program,School of Life Sciences,Tsinghua University,100084,Beijing,China [4]School of Biopharmacy,China Pharmaceutical University,211198,Nanjing,China [5]Chinese Institute for Brain Research,Beijing(CIBR),102206,Beijing,China [6]Chinese Academy of Medical Sciences&Peking Union Medical College,100730,Beijing,China [7]Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program,Academy for Advanced Interdisciplinary Studies,Peking University,100871,Beijing,China [8]Arthritis Clinic and Research Center,Peking University People’s Hospital,Peking University,100044,Beijing,China [9]Musculoskeletal Research Laboratory,Department of Orthopaedics&Traumatology&Li Ka Shing Institute of Health Sciences,Faculty of Medicine,The Chinese University of Hong Kong,999077,Shatin,Hong Kong SAR,PR China [10]Department of Radiology,Beijing Jishuitan Hospital,Capital Medical University,National Center for Orthopaedics,100035,Beijing,China [11]Department of Dermatology,University Hospital Essen&German Cancer Consortium,Partner Site,Essen,45147,Germany [12]Department of Molecular Metabolism,Harvard T.H.Chan School of Public Health,Boston,MA,02115,USA [13]Tsinghua Institute of Multidisciplinary Biomedical Research,Tsinghua University,100084,Beijing,China

出　　处：《Bone Research》2025年第1期104-120,共17页骨研究(英文版)

基　　金：National Natural Science Foundation of China(grant number 82272563 to B.S.);National Science and Technology Major Project of the Ministry of Science and Technology of China(grant number 2023ZD0501202 to B.S.);institutional grants allocated to the National Institute of Biological Sciences,Beijing(NIBS)from the Chinese Ministry of Science and Technology,Beijing Municipal Commission of Science and Technology,and Tsinghua University;the support from China Pharmaceutical University(grant number 3150140001 to S.F.);National Natural Science Foundation of China(grant numbers 82203653 to S.F.,82371957 to L.W.,and 82371956 to X.C.);Beijing Municipal Public Welfare Development and Reform Pilot Project for Medical Research Institutes(grant number JYY2023-8 to X.C.);Research Grants Council of University Grants Committee Hong Kong(grant numbers 14113723,14108720,14121721,14202920,N_CUHK472/22,C7030-18G,T13-402/17-N,and AoE/M-402/20)。

摘　　要：Tissue clearing combined with high-resolution confocal imaging is a cutting-edge approach for dissecting the three-dimensional(3D)architecture of tissues and deciphering cellular spatial interactions under physiological and pathological conditions.Deciphering the spatial interaction of leptin receptor-expressing(LepR^(+))stromal cells with other compartments in the bone marrow is crucial for a deeper understanding of the stem cell niche and the skeletal tissue.In this study,we introduce an optimized protocol for the 3D analysis of skeletal tissues,enabling the visualization of hematopoietic and stromal cells,especially LepR+stromal cells,within optically cleared bone hemisections.Our method preserves the 3D tissue architecture and is extendable to other hematopoietic sites such as calvaria and vertebrae.The protocol entails tissue fixation,decalcification,and cryosectioning to reveal the marrow cavity.Completed within approximately 12 days,this process yields highly transparent tissues that maintain genetically encoded or antibody-stained fluorescent signals.The bone hemisections are compatible with diverse antibody labeling strategies.Confocal microscopy of these transparent samples allows for qualitative and quantitative image analysis using Aivia or Bitplane Imaris software,assessing a spectrum of parameters.With proper storage,the fluorescent signal in the stained and cleared bone hemisections remains intact for at least 2–3 months.This protocol is robust,straightforward to implement,and highly reproducible,offering a valuable tool for tissue architecture and cellular interaction studies.

关 键 词：TRANSPARENT SECTIONS STRAIGHT 

分 类 号：R73[医药卫生—肿瘤]