Lsm 700 laser confocal microscope

For cell proliferation assay, both primary cardiomyocytes and H9c2 cell line were incubated in 24 well plates with different treatments. DNA synthesis was then analyzed by 5-Ethynyl-2’-deoxyuridine (EdU) labeling, using Cell-Light EdU Apollo567 In Vitro Imaging Kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. At least 7 images were randomly taken per well using a Zeiss LSM 700 laser confocal microscope (Carl Zeiss). The population of EdU⁺ cells was determined by counting at least 500 cells per well. The EdU⁺ cells were quantified as the percentage of total cells. Moreover, proliferation of primary cardiomyocytes was also performed by Ki67 (Abcam, ab16667, 1:250) staining. In addition, cell numbers were analyzed using the Enhanced Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, China) according to the manufacturer’s instructions, as previously described (Zhou et al., 2018 (link)).

Hearts were embedded in paraffin and cut in 5 μm sections, deparaffinized, rehydrated and antigen retrieval. Sections were permeabilized with 0.5% Triton X-100/PBS and then blocked with 5% goat serum (Jackson ImmunoResearch Laboratories, USA) for 1 hr at room temperature, and incubated with primary antibodies overnight at 4℃. After washing with PBS, sections were incubated with corresponding secondary antibodies conjugated to fluorescence for 1 hr at room temperature, followed by counterstaining with DAPI (Sigma). Primary antibodies are as follows: anti-Mettl3 (Abcam, ab195352, 1:500), anti-m⁶A (Synaptic Systems, 202111, 1:100), anti-Ki67 (Abcam, ab16667, 1:250), anti-phospho-Histone H3 (pH3) (CST, #53348 S, 1:400), anti-aurora kinase B (AurkB) (Abcam, ab2254, 1:200,) anti-GFP (Proteintech, 50430–2-AP, 1:200), and anti-Cardiac Troponin T (cTnT) (ThermoFisher, MA512960, 1:200). Secondary antibodies used are following: Alexa Fluor 488 goat anti-mouse or anti-rabbit IgG (Jackson ImmunoLabs, 115-545-071 or 111-545-003, 1:200), and Cy3-conjugated Affinipure Goat anti-mouse or anti-rabbit IgG (Proteintech, SA00009-1 or SA00009-2, 1:300). The slides were imaged with fluorescence microscope (Leica Microsystems) or Zeiss LSM 700 laser confocal microscope (Carl Zeiss).

For cell proliferation assay, NCTC1469 cells were incubated for 48 h in 24 well plates with different treatments. DNA synthesis was then analyzed by 5-Ethynyl-2'-deoxyuridine (EdU) labeling, using Cell-Light™ EdU Apollo®567 In Vitro Imaging Kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. At least seven images were randomly taken per well using a Zeiss LSM 700 laser confocal microscope (Carl Zeiss). The population of EdU⁺ cells was determined by counting at least 500 cells per well. The EdU⁺ cells were quantified as the percentage of total cells. In addition, cell proliferation was also analyzed by evaluating cell count using the Enhanced Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, China) according to the manufacturer’s instructions.

The morphologies of the electrospun fibers were examined with a high-resolution scanning electron microscopy (SEM) (FEI, Nova 600 NanoSEM). The fibers were placed directly into the SEM chamber without any metal sputtering or coating. All the images were captured using a secondary electron detector with an acceleration voltage of 5 kV under low vacuum conditions.
The core-shell structure of the SF/PLCL fibers was characterized by Transmission electron microscopy (TEM, Tecnai G2 Spirit, FEI Co.). The specimen was prepared by direct deposition of the electrospun fibers onto a carbon film coated copper grid (Ted Pella, Inc., Redding, CA). Images of SF/PLCL fibers were obtained at 120 kV.
To confirm the presences and distribution of TetramethylrhodamineBSA in the core of fibers and FITC-BSA in the shell of fibers, observations of Silk-TetramethylrhodamineBSA/PLCL-PEO-FITC-BSA fibers deposited on microscope glass slides were performed on a Zeiss LSM 700 laser confocal microscope (Carl Zeiss Micro-Imaging GmbH, Germany) and EVOS FL Auto Cell imaging system (Life Technologies, Carlsbad, CA, USA).

Cellular morphology was visualized at day 28 using fluorescence microscopy. Briefly, cells and cell-laden constructs were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for 15 min at room temperature (RT). After rinsing with PBS for three times, the samples were placed in a permeabilization solution with 0.5% (v/v) Triton X-100 for 15 min and rinsed again with fresh PBS for three times. The cells and constructs were then blocked with 1% BSA in PBS for 1 hour at RT. Immunostaining with primary antibodies mouse monoclonal FSP1 (Abcam, Cambridge, UK) (1:400) and Alexa Fluor 488-conjugated donkey anti-mouse IgG (Life technologies, Carlsbad, CA) (1:1000) was performed at 4°C with gently shaking for overnight, then counterstained with Hoechst 33258 (Life technologies, Carlsbad, CA) to visualize the fibroblasts and nuclei, respectively. Negative control was used with mouse isotype IgG (DAKO, Denmark) antibody. The cells and cell-laden constructs were visualized using a Zeiss LSM 700 laser confocal microscope (Carl Zeiss Micro-Imaging GmbH, Germany).

The putative plastid transit peptides of DXS isoforms were predicted using TargetP (Emanuelsson et al., 2000 (link)). Fragments of AaDXS genes encoding transit peptides were amplified by PCR using KOD plus (TOYOBO, Japan). Subsequently, all fragments harboring SacI and SalI restriction sites were inserted into the multiple cloning site of pCAMBIA1300-green fluorescent protein (GFP) in-frame with the coding sequence of the GFP gene. Approximately, 20 μg of each plasmid was introduced into mesophyll protoplasts of tobacco (Nicotiana tabacum) using polyethylene glycol-mediated transformation (Yoo et al., 2007 (link)). GFP fluorescence and chlorophyll autofluorescence were observed using ZEISS LSM700 laser confocal microscope (ZEISS, Germany) at excitation wavelengths of 488 and 555 nm, respectively.