Developer xd 1

All the fluorescence stainings were acquired in a confocal high-resolution microscope Leica TCS-SP5 (AOBS). Type, magnification, and numerical aperture of the objective lenses: 40 × 1.25 oil UV plan apochromat; 63 × 1.4 oil UV plan apochromat. Temperature: room temperature. Imaging medium: Prolong Antifade Kit (Invitrogen). Fluorochromes: AlexaFluor 488, AlexaFluor 555/568, AlexaFluor 647 (Invitrogen). Photomultiplier tubes (PMT). Stacks were taken with a step size of 0.8 Μm and maximum projected by the LAS AF (Leica) software for analysis.
Analysis of fluorescence intensities of all the staining that were analyzed was performed with Definiens Developer XD1.2 or XD 1.5 software (Definiens). Nuclei were segmented with cellenger and developer algorithms. Intensity results were exported with a developer ruleset into Excel.

EPCs were prepared and fixed with 10% paraformaldehyde for 10 min at room temperature. Apoptosis of EPCs as analyzed using TUNEL assays (DeadEnd™ Colorimetric Tunel System; Promega Corporation) according to the manufacturer's instructions. EPCs (1x10⁵) were incubated TUNEL solution at 37˚C for 1 h. Cells were washed with PBS three times for 5 min at 37˚C followed by incubated with 5% DAPI (Sigma-Aldrich; Merck KGaA) for 15 min at 37˚C in a dark wet box. Finally, images in three fields were captured using a ZEISS LSM 510 confocal microscope at 488 nm. The apoptosis rate was calculated using Developer XD 1.2 (Definiens AG).

Apoptosis of hippocampal pyramidal neurons were analyzed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (DeadEnd™ Colorimetric Tunel System; Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. Hippocampal pyramidal neurons (1×10⁵) were incubated TUNE (DeadEnd™ Colorimetric Tunel System; Promega Corporation). Cells were washed with PBST (Sigma-Aldrich) three times for 5 min at 37°C followed by incubated with 5% DPAI (Sigma-Aldrich; Merck KGaA) for 15 min at 37°C. Finally, images were captured with a ZEISS LSM 510 confocal microscope at 488 nm. The apoptosis rate was calculated by using the software of Developer XD 1.2 (Definiens AG, Munich, Germany).

Apoptotic rates of myocardial tissue or myocardial cells were analyzed using TUNEL assay (DeadEnd™ Colorimetric Tunel System; Promega Corporation) according to the manufacturer's instructions. For myocardial tissues, tissues were fixed with 5% paraformaldehyde for 12 h at room temperature. 5-µm-thick sections were fixed with 4% paraformaldehyde for 30 min at 37°C and dehydrated in gradient concentrations of ethanol. Tissue sections or myocardial cells (1×10⁴ cells/well) were incubated with TUNEL reagent for 30 min at 37°C. Cells were washed with PBS three times for 5 min at 37°C, followed by incubation with 5% DAPI (Sigma-Aldrich; Merck KGaA) for 15 min at 37°C. Images were captured using a ZEISS LSM 510 confocal microscope using 488 nm excitation fixed in Antifade Mounting Medium. The apoptotic rate was calculated using the Developer XD 1.2 software (Definiens AG).

TUNEL analysis was conducted using an In Situ Cell Death Detection kit (DeadEnd™ Colorimetric Tunel System; Promega Corporation). For lung tissue, tissue sections (4 µm) were deparaffinized using xylene, rehydrated in graded ethanol, and rehydrated for 3 min. The sections were then incubated with TUNEL (DeadEnd™ Colorimetric Tunel System; Promega Corporation ) for 2 h at 37˚C according to the manufacturer's protocol. For MBECs, cells were treated with 4% paraformaldehyde for 15 min at room temperature. Cells were then washed with PBST three times at room temperature and incubated with TUNEL (DeadEnd™ Colorimetric Tunel System, Promega) for 1 h at 37˚C according to the manufacturer's protocol. Cells were washed with PBS three times at room temperature and then incubated with 5% DAPI (Sigma-Aldrich; Merck KGaA) under Antifade mounting medium (cat. no. P0126; Beyotime Institute of Biotechnology) for 30 min at 37˚C. Finally, images of sections and cells were captured in six fields of view with a ZEISS LSM 510 confocal microscope at 488 nm at a magnification of x100. The apoptosis rate was measured using Developer XD 1.2 software (Definiens AG).

TUNEL assay was used to determine the percentage of cell apoptosis in bone tissue and BMSCs. For bone tissue, three specimens in each group were deparaffinized and stained with TUNEL (Roche Diagnostics) following the manufacturer's instructions. For BMSCs, miR-NC inhibitor-transfected or miR-155 inhibitor-transfected cells were seeded in 6-well plates (1×10⁴ cells/well) and cultured with or without 50 mM puerarin for 144 h. Cells were immersed in 50 μl TUNEL reaction fluid in a humid environment at 37°C for 1 h. After washing with PBS three times, the cells were incubated with DAPI at 37°C for 30 min. Finally, the samples were washed with PBS three times and then captured at a magnification of ×100 with a Leica DM 4000 microscope. The apoptosis rate was calculated using Developer XD 1.2 software (Definiens AG).