At 48 h after transfection with siRNAs, FRET probes for SNAP-23 were expressed in J774 cells together with the Myc vector or Myc-tagged syntaxin11 using XtremeGENE HP DNA Transfection Reagent (Roche Diagnostics K.K., Tokyo, Japan) according to the manufacturer’s instructions. At 20 h after FRET probe transfection, the cells were incubated in the presence or absence of 1 µg/ml LPS for 1 h. The LPS-containing medium was completely removed, and the cells were incubated in the culture medium without LPS for 4 h before analysis. Fluorescence spectra of the probes on the plasma membranes of live cells were obtained using the LSM780meta laser-scanning microscope (Carl Zeiss) at an excitation wavelength of 458 nm. Before measurements were taken, the dynamic range at each wavelength was calibrated using a standard solution according to the manufacturer’s instructions. Analysis of the spectrum with a fluorescence intensity of ∼250 arbitrary units at 503 nm was performed using the LSM780meta microscope. FRET efficiency is represented as the 582/503-nm emission ratio.
Lsm 780 meta laser scanning microscope
Manufactured by Zeiss
The LSM 780 META is a laser scanning microscope designed for advanced imaging applications. It features a highly versatile optical configuration, enabling the capture of high-resolution images across a wide range of samples. The microscope utilizes innovative laser technology and sensitive detectors to provide detailed information about the structure and composition of specimens.
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4 protocols using lsm 780 meta laser scanning microscope
1
SNAP-23 FRET Analysis in Macrophages
2
Mitochondrial P-P66Shc Imaging in HUVECs
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Initially, various groups of HUVECs were prepared and fixed, then after antigen blocking with 2% BSA, the MitoTracker Red (1:1000 dilution ) was used to label mitochondria, then the diluted primary antibody against P-P66Shc, and secondary antibody conjugated with FITC were incubated. An LSM 780 META laser scanning microscope (Zeiss, NY) was used to obtain IF images, and Image J software was used to calculate the fluorescence intensity .
3
Mitochondrial ROS Evaluation in HUVECs
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MitoSOX staining was used to evaluate mitochondrial ROS content in HUVECs. An LSM 780 META laser scanning microscope (Zeiss, NY) was used to obtain MitoSOX images, and then Image J software was used to analysis the MitoSOX fluorescence intensity.
4
Assessing Intracellular ROS and Protein Expression
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Dihydroethidium (1μM, DHE, Sigma-Aldrich) were used to assess intracellular ROS production in 4-mmthick kidney cryostat sections as described previously (32) . The mean uorescence intensity (MFI) of DHE was calculated by ImageJ. For immuno uorescence assay, the cells or frozen sections were xed with 4% paraformaldehyde for 10 min at room temperature. Then, the sections were blocked with 0.1%TritonX-100 and 5%BSA mixture for 60 min at room temperature and then incubated with primary antibody overnight at 4°C. Later incubated secondary antibodies conjugated with Alexa Fluor 488 (green) or 594 (red) for 1 hour. These cells or slides were counterstained with 4′,6-diamidino-2 phenylindole (DAPI) and their uorescent signals were visualized using an LSM 780 META laser scanning microscope (Zeiss, Thornwood, NY). The MFI of p-STAT3 and C/EBP-β was calculated by ImageJ.
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