Immunostaining
Description
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.