Cultured MCF-7 cells (5,000 cells per chamber) were plated onto eight-chamber slides (Lab-tek) and treated with BPA/BPS for 48 h. At the indicated times, the MCF-7 cells were first washed with PBS and then fixed with 4% paraformaldehyde solution for 15 min, followed by another PBS wash. After permeabilization with 0.1% Triton X-100 in PBS for 10 min, the MCF-7 cells were incubated with 4 N HCl for 15 min at RT, rinsed with distilled water, and placed in Tris–HCl (pH 8.5) for 10 min. The MCF-7 cells were washed with PBS and incubated in a blocking solution [3% goat serum in PBS-Tween (PBST; Catalog No. 28352, Pierce, Thermo Scientific)] for 1 h at RT. The MCF-7 cells were then treated with diluted (1:1,000) anti- rabbit polyclonal antibody (Active Motif) overnight at 4°C. Finally, the slides were washed with PBST solution and incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG secondary antibody at the dilution of 1:2,000 for 1 h at RT, followed by application of DAPI ( in PBS) for 10 min to stain the nuclei. Fluorescence imaging was collected by confocal fluorescence microscopy (Leica).
Confocal fluorescence microscopy
Manufactured by Leica camera
Sourced in Germany, Switzerland
Confocal fluorescence microscopy is a specialized imaging technique that allows for the observation of fluorescently labeled samples with high resolution and optical sectioning. The core function of this technology is to capture detailed, three-dimensional images of specimens by selectively focusing on a specific plane within the sample, thereby reducing out-of-focus background noise and improving image clarity.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (3 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (3 mentions)
Exoquick tc, by System Biosciences (3 mentions)
Secondary ab, by Beyotime (2 mentions)
γ h2ax, by Abcam (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Alexa fluor594 labelled secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Ht7800, by Hitachi (1 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc and pi apoptosis detection kit, by BD (1 mentions)
Tunel assay kit, by Roche (1 mentions)
Paraformaldehyde (pfa), by Honeywell (1 mentions)
Tri ton x 100, by Honeywell (1 mentions)
Leica cm1900, by Leica camera (1 mentions)
Saponin, by Merck Group (1 mentions)
Ht7700, by Hitachi (1 mentions)
Click it edu microplate assay, by Thermo Fisher Scientific (1 mentions)
26 protocols using confocal fluorescence microscopy
1
Immunofluorescence Assay for 5hmC in BPA/BPS-Treated MCF-7 Cells
2
Quantifying DNA Damage Response by γH2AX
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Cells were seeded into confocal laser small dishes and harvested at 2, 8, 24h postirradiation with or without foretinib. Cells were subsequently fixed in 4% paraformaldehyde at 4℃ overnight and permeabilized in 0.1% Triton X-100 (Sigma) for 10 min. Thereafter cells were blocked with 5% bovine serum albumin (Gibco) for 1h and incubated with γH2AX antibody overnight at 4℃. Cells were washed with PBS for three times before incubating with an Alexa Fluor 594-conjugated secondary antibody (Jackson ImmunoRearch) for 1h. Cells were treated with DAPI (Beyotime Biotechnology) and visualized using confocal fluorescence microscopy (Leica).
3
Quantifying Tumor Necrosis and TRAIL Expression
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Heavily anesthetized mice were sacrificed by cervical dislocation within 4 weeks of the xenograft. Tumor tissues were collected, photographed, and analyzed. Harvested tumor tissues were fixed with 4% paraformaldehyde and used for frozen sections (thickness, 15 µm) or paraffin sections (thickness, 10 µm). Frozen sections were incubated with anti-human TRAIL antibody (1:200, 27064-1-AP; Proteintech, Wuhan, China), followed by incubation with fluorescent anti-rabbit AF488 secondary antibody (Thermo Fisher Scientific). Nuclei were stained with DAPI (Sigma-Aldrich). CM-Dil-labeled iMSCs and fluorescence staining of TRAIL were visualized and acquired by confocal fluorescence microscopy (Leica, Wetzlar, Germany). Paraffin sections were stained with hematoxylin and eosin for histological examination. Five visual fields for each group were randomly assessed during the statistical analysis of the percentage of necrotic/vacuolated area in the tumor tissue. The area was quantified using ImageJ software.
4
Immunofluorescence Analysis of SLFN5 and Ki-67
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Cells were cultured in cover glass chambers and fixed in 1% paraformaldehyde for 5 min and permeabilized with 1‰ Triton X-100 for 3 min. Cells were treated with 3% goat serum for 30 min at room temperature and incubated with SLFN5 antibody (1 : 100), or Ki-67 antibody (1 : 100) overnight. Cells were then incubated with Alexa Fluor594-labelled secondary antibodies (1 : 1000) (Invitrogen, USA) for 1 hour at room temperature in darkness. Nuclei were counterstained with DAPI, and fluorescent images were captured using confocal fluorescence microscopy (Leica; Germany).
5
Isolation of Primary Mouse Microglia
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All experimental procedures and mouse care were conducted in compliance with the Korea Institute of Science and Technology (KIST) Animal Care and Ethics Committee Guidelines. Primary microglia were isolated from the cortices of 1 or 2 day-old C57BL/6N mouse pups as described previously (Giulian and Baker, 1986 (link)). The brain cortex was removed and cut into 1 mm3 fragments, and tissues were dissociated by mechanical trituration. The cell suspension was filtered through a 40 µm cell strainer, and seeded into T75 culture flask in DMEM with 10% FBS for 2 weeks. Primary microglia were collected by gently shaking, and purity (>95%) was identified with Ionized calcium binding adaptor molecule (Iba)-1 maker using confocal fluorescence microscopy (Leica, Solms, Germany).
6
Visualization of DNA Damage Response
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Cells were seeded a concentration of 5 × 104 into confocal laser small dishes and harvested at 0.5, 1, 6, 24 hours post IR. Cells were subsequently fixed in 4% paraformaldehyde at room temperature for 30 minutes and permeabilized in 0.1% Triton X‐100 (Sigma, Santa Clara, CA, USA) for 15 minutes. The cells were then blocked with 5% BSA (Gibco, NY, USA) for 1 hour and incubated with primary antibody γH2AX (1 μg/mL; Abcam, Cambridge, Cambridgeshire, UK) overnight at 4°C. Cells were washed in TBST 3 times every 5 minutes before incubating with a secondary Ab (Beyotime Biotechnology) for 1 hour. Cells were treated with 2 μg/mL DAPI (Beyotime Biotechnology) for 5 minutes and then visualized using confocal fluorescence microscopy (Leica, Frankfurt, Germany).
7
Quantifying DNA Damage Response with γH2AX
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Cells were seeded a concentration of 5 × 104 into confocal laser small dishes and harvested at 2 hours post IR. Cells were subsequently fixed in 4% paraformaldehyde at room temperature for 30 minutes and permeabilized in 0.1% Triton X-100 (Sigma, Santa Clara, CA, USA) for 15 minutes. The cells were then blocked with 5% BSA (Gibco, NY, USA) for 1 hour and incubated with primary antibody γH2AX (1 μg/mL; Abcam, Cambridge, Cambridgeshire, UK) overnight at 4°C. Cells were washed in Tris-Buffered Saline Tween-20 (TBST) 3 times every 5 minutes before incubating with a secondary Ab (Beyotime Biotechnology) for 1 hour. Cells were treated with 2 μg/mL DAPI (Beyotime Biotechnology) for 5 minutes and then visualized using confocal fluorescence microscopy (Leica, Frankfurt, Germany).
8
Immunofluorescence Staining Protocol
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After dewaxing, samples were incubated with 10 mM citric acid (pH6.0) for antigen repair and blocked with goat serum for 2 hours. Following primary antibody staining, PBS was washed and incubated with fluorescent secondary antibody for 1 hour, then washed with phosphate-buffered saline (PBS). Then, we repeated the above procedure for the second primary antibody staining. Finally, the tissue slices were mounted with medium containing 4’,6-diamidino-2-phenylindole (DAPI). Stained slides were observed by using confocal fluorescence microscopy (Leica, Germany).
9
Immunocytochemistry of Cardiac Fibroblasts
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Six-well plates were prepared with coverslips, and each coverslip was seeded with 200,000 cardiac fibroblasts. The coverslips were carefully collected and subjected to three washes with PBS buffer. Next, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min. After thorough rinsing with PBS buffer, permeabilization was carried out using a solution containing 0.5% Triton-100 at room temperature for 20 min. Subsequently, the coverslips were blocked with a solution containing 5% BSA at room temperature for 1 h. Following the blocking step, the coverslips were incubated overnight at 4 °C with the primary antibody, followed by incubation at room temperature for 1 h with the corresponding secondary antibody. Finally, the coverslips were mounted on glass slides using a mounting medium containing DAPI. Confocal fluorescence microscopy (Leica) was utilized for the visualization and capture of fluorescent images.
10
Quantifying Angiogenesis in Ischemic Muscle
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Paraffin-embedded sections (6 μm) of ischemic hind limb skeletal muscle from each group were subjected to Immunofluorescence (IF) staining. The cells were rinsed with PBS 3 times for 5 min each and then blocked with 10% goat serum at 37°C for 1 h. Next, the cells were incubated with an anti-CD31 (AF3628, R&D) antibody at 4°C overnight. Sections were washed three times with PBS and labeled with a secondary antibody (Invitrogen) at 37°C for 1 h. DAPI (Invitrogen) was used to stain the nuclei. Sections were imaged by confocal fluorescence microscopy (Leica, Germany).
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