After transfection, cells were fixed with 4% paraformaldehyde for 30 min. Then, the cells were added with TUNEL reagent (Roche, Basel, Switzerland) at 37°C for 1 h. The nuclei were visualized with DAPI solution (Beyotime, Shanghai, China). After washing with PBS, the cells were detected using a florescent microscope (Olympus, Tokyo, Japan).
Florescent microscope
Manufactured by Olympus
Sourced in Japan
The Olympus Fluorescent Microscope is a scientific instrument designed to visualize and analyze fluorescent-labeled samples. It utilizes specific wavelengths of light to excite fluorescent dyes or proteins within the specimen, allowing for the observation of cellular structures, molecular interactions, and other fluorescent-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Dapi solution, by Beyotime (1 mentions)
Tunel reagent, by Roche (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Tunel kit, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Tunel staining, by Beyotime (1 mentions)
Tunel apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Fluoromount media, by Merck Group (1 mentions)
Ab187339, by Abcam (1 mentions)
Ab32362, by Abcam (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Mab4470, by Novus Biologicals (1 mentions)
9 protocols using florescent microscope
1
TUNEL Assay for Apoptosis Detection
2
Apoptosis Assessment of Ovarian Granulosa Cells
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The apoptosis of ovarian granulosa cells in D-gal-induced mice was assessed by TUNEL (Beyotime, Shanghai, China) [21 (link)]. In brief, the fixation and permeability of ovarian granulosa cells were conducted with 4% paraformaldehyde (Sigma-Aldrich) and 0.25% Triton‐X 100 (Sigma-Aldrich), respectively. Then, the cells were washed with phosphate buffered saline (PBS), after which DAPI was used to counterstain the nucleus for 10 min. Finally, the positive apoptotic cells were observed under a florescent microscope (Olympus, Tokyo, Japan).
3
Immunofluorescence Assay in 293T Cells
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The procedure of IF assay was carried out as previously described (Xin et al., 2019 (link)). In brief, 293T cells were seeded on glass slides in six-well plates, fixed in 4% formaldehyde, permeabilized with 0.5% Triton X-100, and then blocked with fresh 10% normal goat serum for 1 h. The cells were incubated with primary antibodies at 4 °C overnight. The secondary antibodies were incubated for 1 h at 37 °C. Subsequently, the cells were washed with PBS and applied with Hoechst 33342. Images were acquired with a florescent microscope (Olympus, Tokyo, Japan).
4
Apoptosis Assessment in HK-2 Cells
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In short, HK-2 cells were cultured with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100 and blocked with 5% bovine serum album (BSA). Then, cells were stained with TUNEL reagent for 60 min and counterstained with DAPI (1 μg/ml) for 10 min in the dark, followed by observation under a florescent microscope (Olympus, Tokyo, Japan). The apoptotic rate was determined by calculating the percentage of the number of positive apoptotic cells in the total number of nuclei.
5
TUNEL Assay for Apoptosis in HCT116 Cells
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The apoptosis of HCT116 cells was determined using a TUNEL kit (Beyotime Institute of Biotechnology, Shanghai, China). HCT116 cells harvested in the logarithmic growth phase were injected into 48-well plates at a density of 1×104 cells/mL and then treated as aforementioned. Subsequently, HCT116 cells were subjected to 4% paraformaldehyde (Beyotime Institute of Biotechnology) fixation at room temperature for 30 minutes and 0.25% Triton X-100 (Beyotime Institute of Biotechnology) permeation at room temperature for 5 minutes. Next, HCT116 cells were rinsed with phosphate-buffered saline (PBS) twice and then incubated with 50 µL reaction solution for 1 hour as per the standard protocol, after which 0.1 µg/mL 4',6-diamidino-2-phenylindole (DAPI) was applied for the staining of cell nuclei at room temperature for 10 minutes. Finally, apoptotic cells in 5 randomly selected fields were photographed under a florescent microscope (Olympus, Tokyo, Japan).
6
Apoptosis Assessment of BV2 Cells
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The apoptosis level of BV2 cells was assessed using TUNEL staining (Beyotime Institute of Biotechnology). Paraformaldehyde (4%) and Triton X-100 (0.5%) was used to treat BV2 cells for 15 and 20 min respectively at room temperature. Subsequently, the cells were labeled with TUNEL for 1 h at 37 ˚C. The counterstaining of cells with 1 µg/ml DAPI was performed at 37˚C for 30 min in the dark. The observation of positive cells in five randomly selected fields was performed under a florescent microscope (Olympus Corporation).
7
TUNEL Assay for Apoptosis Detection
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For detecting apoptosis, the spinal cord tissue sections previously generated were submitted for TUNEL staining using a TUNEL apoptosis kit (ThermoFisher USA). The tissue sections were dipped into TUNEL reagent for 60 min, after this the nuclei were stained with DAPI solution (1 μg/ml) for 10 min and then the sections were immersed in Fluoromount media (SigmaAldrich USA). The positive cells were quantified in 10 random field in each slide using florescent microscope (Olympus), the apoptotic cells as well as total cells were counted.
8
Apoptosis Imaging by AO/PI Staining
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Based on cytotoxicity studies, wells were prepared with the same concentrations of substances for imaging of substance-based inhibition and apoptosis of cells with florescent dying and imaging was made daily. 50:50 rated Acridine Orange/Propidium Iodide (AO/PI) florescent dye mixture was used for imaging. For this purpose, medium cultures in wells were collected and each well was washed with 10 × Phosphate-buffered saline (PBS). 100 μL AO/PI was treated to cells in washed-wells for 20 s. After collecting dye, wells were washed with PBS in 10 s and imaged with florescent microscope (Olympus, Waltham).
9
Immunofluorescence Assay for Muscle Markers
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Cells were fixed with 4% PFA, and blocked with 10% FBS, followed by incubation with anti-Pax7 antibody (ab187339, abcam, 1:300), anti-desmin antibody (ab32362, abcam, 1:500) and anti-Myosin Heavy Chain Antibody (MF20) antibody (Novus, MAB4470, 1:200) respectively at 4°C overnight and secondary antibody conjugated to Alexa Fluor 594 or Alexa Fluor 488 (Life Technologies) at room temperature for 1h. Images were taken by a florescent microscope (Olympus, Japan).
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