Cells were seeded on poly-D-lysine coated overslips were transfected with Myc-GLUT4-mCherry plasmid by electrotransfection. Cells were harvested and fixed with 4% paraformaldehyde for 10 min, then washed with cold PBS two times and blocked with PBS containing 5% donkey serum for 45 min at room temperature. Cells were then incubated with primary rabbit anti-Myc antibody overnight at 4 °C, followed by Alexa Fluor 488-conjugated Donkey anti-rabbit secondary antibody at least 1 h at room temperature. After washing, the overslips were mounted with DAKO mounting medium. Mounted samples were subjected to confocal imagining using a Zeiss LSM710 confocal laser microscope system with a x63 NA/1.40 CFI Plan APO VC oil-immersion objective.
Lsm 710 confocal laser microscope system
Manufactured by Zeiss
Sourced in Germany
The LSM 710 is a confocal laser microscope system manufactured by Zeiss. It is designed for high-resolution imaging and analysis of biological and materials samples. The system utilizes laser excitation and confocal optics to capture detailed, three-dimensional images with improved contrast and resolution compared to traditional wide-field microscopy.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Antifade mounting medium, by Beyotime (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
T9284, by Merck Group (1 mentions)
P6148, by Merck Group (1 mentions)
D8417, by Merck Group (1 mentions)
Af0246, by Beyotime (1 mentions)
A0562, by Beyotime (1 mentions)
A0568, by Beyotime (1 mentions)
Cm1950, by Leica (1 mentions)
Dapi solution, by Solarbio (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Triton x 100, by Solarbio (1 mentions)
4 6 diamidino 2 phenylindole dapi staining solution, by Solarbio (1 mentions)
Jc1 staining solution, by Yeasen (1 mentions)
Mitotracker red cmxros, by Yeasen (1 mentions)
11 protocols using lsm 710 confocal laser microscope system
1
Visualization of Myc-GLUT4-mCherry in Cells
2
Immunofluorescence Staining of Hepatocytes
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For these assays, 14 mm round cover slides were placed in each well in a 24-well plate for hepatocytes to adhere to the wall. The cells were fixed with 4% paraformaldehyde (300 μL/well, P6148, Sigma-Aldrich) for 15 min, washed with PBS 3 times (5 min each), and perforated with 0.3% Triton X-100 (500 μL/well, T9284, Sigma-Aldrich) for 15 min to improve cell permeability. The cells were washed again with PBS three times, and then the sealant (3% BSA) was added to seal the samples in the incubator at 37 °C for 1 h. Then, primary antibodies p65 (1:100, AF0246, Beyotime), STIM1 (1:200, AF2614, Beyotime), Orai1 (1:200, 66223-1-Ig, ProteinTech), and Calnexin (1:100; AC019, Beyotime) were added, and the mixture was incubated overnight at 4 °C. After the cells were washed with PBS three times, the fluorescent secondary antibodies (1:500, A0562, and A0568, Beyotime) were added and incubated at 37 °C for 1 h without light. After three washes with PBS, the nuclei were stained with a DAPI solution (D8417; Sigma-Aldrich), and a round glass cover was fixed on the glass slide after washing for three times in the dark. The cells were observed by an LSM 710 confocal laser microscope system (Zeiss, Oberkochen, Germany).
3
Immunofluorescence Staining of Paraffin Sections
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The prepared paraffin sections were permeated with 0.5% Triton X-100 at room temperature for 10 min. The sections were then treated with citrate buffer for antigenic repair, followed by incubation with 3% H2O2 for 30 min and closed with 1% BSA for 30 min at room temperature. The sections were incubated with the corresponding primary antibody at 4 °C overnight. On the second day, the sections were washed with PBS and incubated with the corresponding fluorescent secondary antibody. After cleaning with PBS, the nuclei were stained with a DAPI solution under the condition of avoiding light. After cleaning again with PBS, anti-fluorescence quenching was added to seal the tablet. The fluorescent images were visualized by a LSM 710 confocal laser microscope system (Zeiss, Oberkochen, Germany). All antibodies used in this part were listed in Supplementary Table S2 .
4
Measuring Liver ROS with DHE Assay
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The ROS was detected with a dihydroethidium (DHE) kit supplied by Yeasen Biotech (Shanghai, China). Briefly, the frozen liver samples were cut into slices with a cryostat (CM1950, Leica, Germany). Each frozen slice was incubated with 200 μL of 10 μM DHE solution at 37°C for 60 min. After washing with PBS 3 times, the fluorescence was detected and quantitated by LSM 710 confocal laser microscope system (Zeiss, Oberkochen, Germany).
5
Immunofluorescence Staining of BMECs
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BMECs were seeded onto the circular coverslips in 12-well plates. When cells grew 70–80% confluence, they were treated according to the experimental design. Cells were fixed with 500 μL/well 4% paraformaldehyde (Sigma-Aldrich, Burlington, MA, USA) for 20 min. The cell morphology on the coverslips was checked under a microscope. Cells were incubated with 0.3% Triton X-100 (500 μL/well, T9284, Solarbio Life Sciences, Beijing, China) for 15 min at RT to increase permeability. Blocking agent (1 × PBS/5% BSA/0.3% Triton X-100) was used to block cells at RT for 1 h. The primary antibodies, which are listed in Supplementary Table S2 , were diluted in antibody buffer (1 × PBS/1% BSA/0.3% Triton X-100) and were used to incubated cells at 4 °C overnight. Then, cells were incubated with the FITC-labeled secondary antibodies, which are listed in Supplementary Table S2 , and counterstained with DAPI (D8417, Sigma Aldrich, Burlington, MA, USA) to stain the nucleus for 5 min. The coverslips were fixed on glass slides by Antifade Mounting Medium (P0128, Beyotime, Shanghai, China). The fluorescent images of each target protein and nucleus inside the cells were visualized by an LSM 710 confocal laser microscope system (Zeiss, Oberkochen, Germany).
6
Immunofluorescence Assay Protocol for BMECs
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BMECs were seeded into 12-well cell plates which were equipped with sterile cell coverslips. After corresponding cell treatment, cellular immunofluorescence assay was performed as described next. The cells were fixed with 4% paraformaldehyde for 20 min after 1 × PBS wash and then incubated with 0.3% Triton X-100 for 15 min to increase cellular membrane permeability. Then the cells were further blocked with blocking solution containing 5% bovine serum albumin for 30 min followed by incubation overnight at 4 °C with the specific primary antibody. After washing, the coverslips were incubated for 60 min at 37 °C with the corresponding FITC-linked secondary antibody. Antibodies used in the cellular immunofluorescence assay are also summarized in the Supplemental Table S2 . Then, the results were visualized using an LSM 710 confocal laser microscope system (Zeiss, Oberkochen, Germany) after the cell nuclear staining with DAPI solution (Cat: C0060, Solarbio Life Sciences, Beijing, China). This was kept gently operating throughout the whole experiment. The experimental conditions, especially the permeabilization time, could be optimized according to the characteristics of the different target proteins.
7
Immunofluorescence Staining Protocol
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After adding coverslips to each well of the 6-well plates, we inoculated 5 × 10 4 cells per well. After the cells were treated as described for the 4 groups, they were washed with 200 μL of 1× PBS 3 times for 5 min each time, gently shaken while washing, fixed with 4% paraformaldehyde for 15 to 20 min, washed 3 times with 1× PBS, and then perforated with 0.25% Triton X-100 (T9284; Sigma-Aldrich, St. Louis, MO) for 15 min. After the perforation solution was discarded, the cells were washed with 1× PBS 3 times, incubated at 37°C for 30 min, and then incubated with the primary antibody (anti-p65, NOD1, p-ERK, or Nrf2, the same as those used in the Western blot analysis) at 4°C overnight. The following day, the cells were incubated at 37°C for 30 min and then washed with 1× PBS 3 times. Next, the cells were incubated with fluoresceinisothiocyanate-conjugated goat anti-rabbit and goat anti-mouse antibodies (1:500, catalog no A0562 and A0568, respectively; Beyotime Biotechnology) at 37°C for 1 h in the dark. Then, the cells were washed with 1× PBS 3 times in the dark, incubated with DAPI (D8417; Sigma-Aldrich) for 5 min in the dark, washed with 1× PBS 3 times in the dark, and then mounted onto slides with 1 drop of antifade mounting medium (P0128; Beyotime Biotechnology). The cells were observed with an LSM 710 confocal laser microscope system (Zeiss).
8
GLUT4 Translocation Quantification
9
Immunofluorescence Staining of Hepatocytes
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Hepatocytes were plated on circular coverslips in the wells of the 24-well microplate. After the indicated treatment, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature. After 3 gentle washes with PBS, the cells were incubated with 0.3% Triton X-100 (Solarbio) for 15 min at room temperature to improve permeability. Subsequently, 3% BSA served as the blocking agent to block the hepatocytes for 1 h at room temperature. Then, the coverslips were incubated with the primary antibodies at 4°C for 16 h. The cells were then incubated with the fluoresceine isothiocyanate (FITC)-or Cy3-labeled secondary antibodies for 1 h at 37°C in the dark. After 3 gentle washes with PBS containing Tween 20 (PBST), 4',6-diamidino-2-phenylindole (DAPI) staining solution (Solarbio) was used to counter-stain nuclei in the dark for 10 min. The coverslips were mounted on glass slides using antifade mounting medium and visualized under a laser scanning microscope (LSM) 710 confocal laser microscope system (Zeiss). The primary and second antibody details are listed in the Supplemental Table S2.
10
Visualizing Mitochondria and Lysosomes in Bovine Hepatocytes
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Bovine hepatocytes were grown on circular coverslips in the 24-well plates. After the indicated incubation, the cells were incubated with 100 nM Mito-tracker red CMXRos (40741ES50, Yeasen) for 15 min, a 50 nM Lyso-tracker red solution (C1046, Beyotime) for 30 min, a 10 μg/mL JC-1 staining solution (40705ES03, Yeasen) for 15 min at 37°C in the dark. After washing with PBS, the cells were observed under a laser scanning microscope (LSM) 710 confocal laser microscope system (Zeiss).
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