Laser scanning confocal microscope

The full-length sequence of AtFDH1 with native promoter was cloned into pMDC107 for GFP expression (AtFDH1-GFP). Stable Arabidopsis transgenic lines for the expression of AtFDH1-GFP were developed by floral dip transformation [48 (link)]. The localization of AtFDH1-GFP in epidermal cells was determined under the confocal laser scanning microscope (NIKON, Japan).
To observe the localization of AtFDH1, Arabidopsis wild-type Col-0 and AtFDH1-GFP expressing (under the control of AtFDH1 promoter) transgenic plants in Col-0 were grown in ½ MS media for four weeks, and AtFDH1-GFP expression in epidermal cells of Arabidopsis was visualized using a confocal laser scanning microscope (NIKON, Japan). The leaf tissues were floated with the bacterial suspension of host pathogen P. syringae pv. maculicola (1×10⁶ CFU/ml) and nonhost pathogen P. syringae pv. tabaci (1×10⁶ CFU/ml). After one hour inoculation, the leaf tissues were washed with distilled water, and localization of FDH1-GFP was observed. For wounding stress, the adaxial epidermal peels from wild-type Col-0 and AtFDH1-GFP expressing transgenic plants were prepared in the MES buffer (10 mM, pH 6.5), and localization of AtFDH1 was imaged under the confocal laser scanning microscope (NIKON, Japan).

For in vivo phagocytosis experiment, CTX-induced TA injury was established as mentioned above while MSCs-ApoEVs were pre-stained by PKH26 (Sigma, USA) according to the manufacturer’s protocol. The PKH26 labeled MSCs-ApoEVs were injected into left TA muscles 3 days after TA injury models were established. The TA muscles were collected and fixed, dehydrated, and embedded in optimal cutting temperature compound (Leica, Germany) 24 h after PKH26 labeled MSCs-ApoEVs injection. For immunofluorescence staining, 10 μm sections were incubated with mouse anti-myosin antibody (R&D, MAB4470) at 1:250 dilution overnight at 4 °C and then incubated with Alexa Fluor 488 labeled secondary antibody (Yeasen, 33112ES60) at 1:500 dilution for 2 h at room temperature and finally stained with Hoechst 33342 (Invitrogen, H21492) for 10 min at room temperature, all operations are carried out in dark environments. The fluorescence images were captured by laser scanning confocal microscope (Nikon, Japan).
For in vitro phagocytosis experiment, PKH26 labeled MSCs-ApoEVs were added to C2C12 myoblasts and incubated for 3 h. After that, C2C12 myoblasts were fixed, permeated, and then stained by 488-conjugated phalloidin (AAT Bioquest, 23115) for 1 h and Hoechst 33342 for 5 min at room temperature. The fluorescence images were captured by laser scanning confocal microscope (Nikon, Japan).