Immunostaining

Histological analysis of whole-mount BM was performed following a slight modification of the previous study. Briefly, femurs and tibias from indicated mice were isolated and fixed for 24 h at room temperature with 4% paraformaldehyde (PFA; Affymetrix, Cleveland, OH, USA) and subsequently bisected along the long axis to expose the BM cavity. Bones were post-fixed for 30 minutes with the same fixation solution stated above and blocked with 10% goat serum (Thermo Fisher Scientific) for 30 minutes. For analysis of anatomical locations of Tregs and HSCs, the slides were successively stained with anti-mouse CD150 (TC15-12F12.2), anti-mouse Connexin 43/GJA1 (CX-1B1), anti-mouse Foxp3 (FJK-16s), PE-conjugated anti-mouse MHC-II (I-A/I-E), biotin-conjugated anti-mouse CD48 (HM48-1), and biotin-conjugated anti-mouse Lineage cocktail (TER-119, RB6-8C5, RA3-6B2, M1/70, 145-2C11) antibodies at 4℃ overnight. Subsequently, the slides were incubated with Pacific Blue Conjugate Streptavidin (Thermo Fisher Scientific) and corresponding fluorescent secondary antibody for 1 h at room temperature. Imaging was performed using a Zeiss LSM800 NLO confocal microscope (Carl Zeiss, Jena, Germany). ZEN software (Carl Zeiss) was used to identify CD150+CD48-Lin- HSCs and Foxp3+ Tregs and the distance between cell centers was calculated. Data were collected in an automated blinded fashion. MHCII+ HSCs were defined as HSCs with relative high fluorescence intensity of MHCII. For analysis of DNA damage in HSCs, cells were stained with PE-conjugated anti-mouse MHC-II (I-A/I-E) and FITC-conjugated anti-mouse phospho-γH2AX (Ser139) at 4℃ overnight. Subsequently, the slides were incubated with DAPI for 5 min at room temperature. Fluorescence intensity of phospho-γH2AX of HSCs with various MHCII expression were compared by ZEN software.

对全-mount BM 进行了组织学分析，其方法基于先前研究的轻微修改。概括而言，选取指定小鼠的股骨和胫骨，在室温下使用4%多聚甲醛（PFA；Affymetrix，克利夫兰，俄亥俄州，美国）固定24小时，随后沿长轴切开以暴露骨髓腔。骨组织在上述固定液中再固定30分钟，并使用10%山羊血清（Thermo Fisher Scientific）封闭30分钟。为了分析Treg和HSCs的解剖位置，将切片依次用抗小鼠CD150（TC15-12F12.2）、抗小鼠连接蛋白43/GJA1（CX-1B1）、抗小鼠Foxp3（FJK-16s）、PE偶联抗小鼠MHC-II（I-A/I-E）、生物素偶联抗小鼠CD48（HM48-1）以及生物素偶联抗小鼠谱系混合抗体（TER-119, RB6-8C5, RA3-6B2, M1/70, 145-2C11）在4℃下过夜染色。随后，将切片与太平洋蓝偶联的链霉亲和素（Thermo Fisher Scientific）和相应的荧光二抗在室温下孵育1小时。使用蔡司LSM800 NLO共聚焦显微镜（卡尔·蔡司，耶拿，德国）进行成像。利用ZEN软件（卡尔·蔡司）识别CD150+CD48-Lin- HSCs和Foxp3+ Tregs，并计算细胞中心的距离。数据以自动化且盲法的方式收集。MHCII+ HSCs被定义为MHCII荧光强度相对较高的HSCs。为了分析HSCs中的DNA损伤，细胞被用PE偶联抗小鼠MHC-II（I-A/I-E）和FITC偶联抗小鼠磷酸化γH2AX（Ser139）在4℃下过夜染色。随后，在室温下用DAPI孵育5分钟。通过ZEN软件比较具有不同MHCII表达水平的HSCs中磷酸化γH2AX的荧光强度。