Immunofluorescence staining was conducted to detect FoxO1 localization after WEE treatment. Briefly, 5 × 104 cells were cultured on chamber slide overnight. The 24 h treatments were followed by insulin (100 nM) stimulation for 20 min; the cells were fixed with formaldehyde in PBS (w:v, 4%) for 15 min and washed with PBS and permeabilized with Triton X-100 (0.25%) during 30 min at 37 °C. Cells were then blocked for 1 h with BSA (2%) and incubated with FoxO1 (1:800) overnight at 4°C, respectively. After washing with cold PBS, cells were incubated with to Alexa Fluor 488-conjugated secondary antibody (1:500) for 1 h at room temperature. Coverslips were mounted with DAPI (YEASEN, Shanghai, China) and visualized in Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan).
A1r eclipse ti confocal microscope
Manufactured by Nikon
Sourced in Japan
The Nikon A1R Eclipse Ti confocal microscope is a high-performance imaging system designed for advanced research applications. It features a modular design, allowing for customization to meet specific experimental requirements. The microscope provides high-resolution imaging, enabling detailed analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Chamber slide, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Yeasen (1 mentions)
Annexin 5 fluorescein isothiocyanate propidium iodide apoptosis detection kit, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Yesen (1 mentions)
Anti 3 nitrotyrosine antibody, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Hoechst 33342, by Solarbio (1 mentions)
Triton x 100, by Merck Group (1 mentions)
9 protocols using a1r eclipse ti confocal microscope
1
Visualizing FoxO1 Localization by Immunofluorescence
2
Immunofluorescence Analysis of Transcription Factor Localization
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Immunofluorescence staining was conducted to detect the NF-κB/p65, AP-1/c-Jun, and IRF3 localization after the RIS treatment. Briefly, with an inoculation volume of 1 mL, 1 × 104 cells were cultured on coverslips (Cat. 177399; Thermo Fisher Scientific) overnight, treated with RIS (200 μg mL−1), and then fixed with formaldehyde in PBS (w/v, 4%) for 15 min. Then, the cells were washed with PBS and permeabilized with Triton X-100 (0.25%) in 30 min at 37 °C and blocked for 1 h with BSA (2%). The cells were then incubated with NF-κB/p65 (1 : 300), c-Jun (1 : 300), and IRF3 (1 : 100) overnight at 4 °C, respectively. After washing with cold PBS, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody (1 : 500) for 1 h at room temperature. The coverslips were mounted using Fluoroshield with DAPI (YESEN, Shanghai, China) and visualized using the Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan).
3
Annexin V-FITC/PI Apoptosis Assay
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Apoptosis was determined using the annexin V-fluorescein isothiocyanate/propidium iodide (PI) apoptosis detection kit (Beyotime Biotechnology) according to the manufacturer’s instructions. Briefly, the cells were cultured in chamber slides (3×104 cells per well), allowed to attach overnight and exposed to various experimental conditions. After treatment for 72 h, the RCEC cells were washed with PBS and incubated with annexin-V labeling solution (containing 10 μL annexin-V and 5 μL PI solution in 200 μL incubation buffer) for 15 min in the dark. Confocal images were acquired on a Nikon A1R Eclipse Ti confocal microscope (Nikon Corporation, Tokyo, Japan).
4
Immunofluorescence Analysis of Transcription Factors
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After the RAW264.7 cells (1 × 104 cells per well) were seeded in chamber slides for 24 h, the cells were washed twice with ice-cold PBS and then fixed with 4% formaldehyde for 15 min at RT. After washing with PBS for three times, the cells were permeabilized by incubation with 400 μL of a 0.25% Triton X-100 solution for 30 min at 37 °C and followed by incubated for 10 min at room temperature. After blocking the cells with 3% BSA for 1 h, the cells were incubated with the polyclonal antibodies for NF-κB/p65, AP-1/c-Jun, and IRF3 diluted at a 2% BSA solution followed by incubation overnight at 4 °C. After being washed 3 times with PBS, the cells were incubated with the Alexa Fluor 488-conjugated secondary antibody for 1 h at RT. The nuclei were stained with DAPI (YESEN, Shanghai, China), and the fluorescence was visualized using the Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan).
5
Quantifying Nitrosative Stress in Rat RCECs
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RCECs were grown on a coated coverslip (3×104 cells per well), allowed to attach overnight and exposed to various experimental conditions. After treatment for 72 h, rat RCECs were fixed in fresh cold 4% paraformaldehyde at 4°C for 30 min and washed three times in PBS. Cells were blocked for 1 h in 5% normal goat serum and incubated with the primary antibody (anti-3-nitrotyrosine antibody 1:100; Abcam, Cambridge, MA, USA) in a moist chamber at 4°C overnight. Following three washes with PBS, the cells were incubated for 1 h at room temperature with a fluorescein-conjugated goat anti-mouse secondary antibody (1:00; Zhong Shan Jin Qiao, Beijing, People’s Republic of China). The coverslips were then mounted onto slides with 4′,6-diamidino-2-phenylindole–containing medium. Images were collected on a Nikon A1R Eclipse Ti confocal microscope (Nikon Corporation) and processed with Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
6
Immunofluorescence Assay for NF-κB, AP-1, and IRF3
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RAW264.7 cells were directly cultured on chamber slides (Thermo Scientific, Waltham, MA, USA) overnight to detect NF-κB/p65, AP-1/c-Jun, and IRF3 localization by immunofluorescence assays. Briefly, after 60 min of stimulation with LPS (1 μg/mL) in the presence or absence of SCL, the cells were washed with cold PBS, and fixed with 4% formaldehyde in PBS for 15 min at room temperature. Subsequently, the cells were permeabilized with 0.25% Triton-x for 30 min at 37 °C and followed by 10 min at room temperature. After blocking the cells with 2% BSA for 1 h, primary antibodies against NF-κB/p65 (1:300), c-Jun (1:300), and IRF3 (1:100), respectively, were incubated overnight at 4 °C. After washing with PBS for 3 times, cells were incubated for 1 h at room temperature with Alexa Fluor 488-conjugated secondary antibody (1:500). After the nuclei were stained with DAPI, and fluorescence was visualized using a Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan).
7
Glucose Uptake Assay in HepG2 Cells
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The glucose uptake was determined as previously reported with some modification (Nie et al., 2017 (link)). Briefly, HepG2 cells (4 × 104 cells/well) were cultured in a chamber slide (Thermo Scientific, Waltham, MA, USA). The 24 h treatments were followed by insulin (100 nM) for 30 min and 0.3 mM 2-NBDG for 45 min incubation at 37°C. The cells were then washed three times by PBS and then incubated with Hoechst 33342 (Solarbio, Beijing, China) for 30 min. After being washed with PBS three times, the cells were incubated with Hank’s solution. Images were obtained using identical acquisition settings on a Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan). The fluorescence intensity and area were analyzed using Image J software (National Institutes of Health [NIH], Bethesda, MD, USA) as previously described (Bankhead, 2014 (link)). The fluorescence intensity and area were normalized with the cell number. Seven images for each group were calculated.
8
Immunofluorescence analysis of GEnCs
9
Fluorescent in situ hybridization of GFP embryos
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Embryos were dissected in DEPC-treated PBS and fixed ON in 4 % PFA in PBS at 4 °C. Afterwards they were washed three times 30 min in DEPC-treated PBS, immersed in 15 % sucrose and snap frozen in Optimal Cutting Temperature (OCT) compound (Richard-Allan Scientific #6502) with liquid nitrogen. The GFP fluorescent in situ hybridisation (ISH) probe was custom designed with the probe designer tool from Stellaris (LGC biosearch technologies). The coding sequence of the pEGFP-N2 plasmid (accession number U57608.1) was used for probe design.
Fluorescent ISH (FISH) was performed according to the manufacturer’s protocol (Stellaris) with the addition of a permeabilisation step with 1 % TritonX100 (Sigma T8787) in PBS. Images were acquired using a Nikon A1R Eclipse Ti confocal microscope.
Fluorescent ISH (FISH) was performed according to the manufacturer’s protocol (Stellaris) with the addition of a permeabilisation step with 1 % TritonX100 (Sigma T8787) in PBS. Images were acquired using a Nikon A1R Eclipse Ti confocal microscope.
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