We added 2 μL GPX2 adenovirus when cells grew to 70% density. After the cells converged, DMEM containing 4% horse serum and 100 units/mL penicillin–streptomycin was used for myogenic differentiation, and the cells were collected after 6 days of induction. Cells were fixed with 4% paraformaldehyde at room temperature for 15 min and permeability with 0.1% Triton X-100 for 10 min. After blocking for 30 min on ice, cell nuclei were stained by incubating the cells in 1 mg/mL DAPI for 5 min at room temperature. Fluorescence was observed with a Motic fluorescence microscope (Motic, Xiamen, China). Since the virus overexpression vector contains green fluorescent protein, which fills the muscle fiber, the muscle differentiation rate and the myotube fusion index were calculated directly after nuclear staining.
Fluorescence microscope
Manufactured by Motic
Sourced in China, Germany, Hong Kong
The Fluorescence Microscope is an advanced imaging tool that utilizes the principles of fluorescence to visualize and analyze samples. It employs specialized light sources and filters to excite fluorescent molecules within the sample, allowing for the detection and study of specific cellular structures, proteins, or other fluorescently labeled components. The Fluorescence Microscope provides high-contrast, high-resolution images that enable researchers to gain valuable insights into the structure and function of biological specimens.
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Lab products found in correlation
Rnai mate, by GenePharma (2 mentions)
Nc shrna plasmid, by GenePharma (2 mentions)
Confocal microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
5 ethynyl 2 deoxyuridine (edu), by RiboBio (2 mentions)
Abn78, by Merck Group (1 mentions)
6 diamidino 2 phenylindole dapi d9542, by Merck Group (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Cell light edu apollo488 in vitro kit, by RiboBio (1 mentions)
Dmem containing edu, by RiboBio (1 mentions)
Ab39398, by Abcam (1 mentions)
Ab177487, by Abcam (1 mentions)
Tunel reaction mixture, by Beyotime (1 mentions)
Goat serum, by ZSGB-BIO (1 mentions)
Anti fluorescence quenching sealing solution, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Alexa fluor 488 555, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 555 647, by Thermo Fisher Scientific (1 mentions)
Anti flna, by Merck Group (1 mentions)
Anti lpar 1, by Abcam (1 mentions)
20 protocols using fluorescence microscope
1
Adenovirus-Mediated Myogenic Differentiation
2
Adenovirus-Induced Myogenic Differentiation
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GPX2 adenovirus (2 μL) was added when cells grew to 70% density. After the cells converged, DMEM/F12 containing 4% horse serum and 100 units/mL penicillin-streptomycin was used for myogenic differentiation. The cells were collected after a certain time of induction. After adding the cell fixing solution (4% paraformaldehyde) and letting it sit at room temperature for 15 minutes, the cells were three times rinsed with PBS for five minutes each. Permeability with 0.5% Triton X-100 for 10 minutes and cells were washed 3 times. Cell nuclear was stained through DAPI for 25 minutes at room temperature. Fluorescence was observed with a Motic fluorescence microscope (Motic, Ximen, China). Because the virus overexpression vector contains green fluorescent protein, which fills the muscle fiber. Therefore, the muscle differentiation index and myotube fusion index can be calculated directly after nuclear staining.
3
Histopathological Analysis of Paraffin-Embedded Tissues
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Fixed tissues that were paraffinized were sectioned at a thickness of 5 μm. HE staining was performed, and histopathological changes were observed using a fluorescence microscope (Motic China Group Co., Ltd.).
4
Quantifying Apoptosis in Peri-Hematoma
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Apoptosis in the peri-hematoma area was detected using the terminal deoxynucleotidyl transferase biotin-mediated dUTP Nick-end labeling (TUNEL) staining kit (DeadEnd Fluorometric TUNEL System, Promega, Madison, WI, USA). Subsequently, the neurons were stained with rabbit anti-neuronal specific nuclear protein (NeuN) (1:1000; ABN78; Sigma-Aldrich). Nuclei were stained with 6-diamidino-2-phenylindole (DAPI) (D9542; Sigma-Aldrich). Images were acquired using a fluorescence microscope (Motic, China). For each group, five random fields were counted to calculate the percentage of apoptotic neurons. The results are presented as the number of TUNEL/NeuN double-positive cells divided by the number of DAPI-stained nuclei counted × 100%.
5
EdU Proliferation Assay in A-498 Cells
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A-498 cells stably expressing shctrl or shRGS20 were seeded on 96-well plates at a density of 100,000 cells/well. After 24 h of culture at 37°C, the cells were incubated at 37°C for 4 h with DMEM containing EdU (50 µM; Guangzhou RiboBio Co., Ltd.). Subsequently, cells were fixed with 4% formaldehyde for 20 min at room temperature, followed by the addition of 2 mg/ml of glycine for 5 min at room temperature. After treatment with 0.5% Triton X-100 at room temperature for 10 min, the cells were washed twice with PBS and treated with 200 µl of 1X Apollo reaction cocktail from a Cell-Light EdU Apollo488 In Vitro kit (Guangzhou RiboBio Co., Ltd.) for 20 min at room temperature, according to the manufacturer's protocol. The nuclear DNA was stained with DAPI (5 µg/ml) for 10 min at room temperature. Images were obtained using a fluorescence microscope (Motic Incorporation, Ltd.; magnification, ×100).
6
Immunofluorescence Detection of p-IGF-1R
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The expression and distribution of p-IGF-1R were detected by immunofluorescence staining. Cells were pre-seeded on the slide and fixed with 4% paraformaldehyde for 15 minutes. After scouring with PBS to remove residual paraformaldehyde, the cells were infiltrated with 0.1% TritonX-100 for 30 minutes to permeate the cell membrane. Then, goat serum was used to block the nonspecific antigen for 15 minutes and spent the night with the anti-p-IGF-1R antibody (ab39398, Abcam) at 4°C. After washing with PBS, the cells were incubated with secondary bodies anti-Rabbit IgG (H + L) for 90 minutes. The nucleus was incubated with DAPI. Finally, the cells were sealed with buffered glycerin and stored away from light for observation under a fluorescence microscope (Motic, Germany).
7
Quantifying Cellular Oxidative Stress
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Cells were plated into 6-well cell culture plates at a density of 4×105 cells/well were treated according to the aforementioned methods. After being washed with PBS twice, the cells were incubated with 2 ml medium containing 10% FBS and 5 µM DHE for 30 min at 37°C in the dark and were then visualized under a fluorescence microscope (Motic Incorporation, Ltd.). Red staining indicating oxidative stress was semi-quantified (ROS-positive staining) using ImageJ software (Version 2.1.0/1.53c).
8
Lentivirus-based NEK6 Knockdown
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A lentivirus-based NEK6-homo-657 RNA plasmid (shNEK6) and an NC-shRNA plasmid were constructed (GenePharma, Shanghai, China). The NEK6 gene was cloned in LV3 (H1/GFP&Puro) plasmids. LV3-NEK6-homo-657 and LV3-shNC plasmids were transfected into 293T cells using RNAi-mate (GenePharma) according to the manufacture's protocol. The cells were harvested 72h after transfection and stable cells were selected and detected by Fluorescence microscope (Motic, China).
9
Apoptosis assessment in rat cortex
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Apoptosis of neurons in the ipsilateral cortex of SD rats was evaluated by terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) and NeuN double immunostaining according to the manufacturer’s protocol. The brain sections from each group were incubated with TUNEL reaction mixture (Beyotime, Shanghai, China) for 1 h at room temperature and then stained with anti-NeuN (ab177487, Abcam, Cambridge, United Kingdom) and DAPI (Wellbio, China). The slides were observed using a fluorescence microscope (Motic, China).
10
Lentiviral Knockdown of NEK6 in 293T Cells
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A lentivirus-based NEK6-homo-657 RNA plasmid (shNEK6) and an NC-shRNA plasmid were constructed (GenePharma, Shanghai, China). The NEK6 gene was cloned in LV3 (H1/GFP&Puro) plasmids. LV3-NEK6-homo-657 and LV3-shNC plasmids were transfected into 293T cells using RNAi-mate (GenePharma) according to the manufacture's protocol. The cells were harvested 72h after transfection and stable cells were selected and detected by Fluorescence microscope (Motic, China).
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