EU (ST2055-50 mg, Beyotime, Shanghai, China) was added to the complete culture medium from a 100 mM stock in DMSO. Gastric cancer cells were cultured with EU for 6 h. The cells were then fixed for 20 min in a 4 % paraformaldehyde suspension and permeabilized with 0.5 % PBS-Triton X-100 for 10 min. The cells were rinsed with TBST and stained for 30 min at room temperature with 100 mM Tris, pH 8.5/1 mM CuSO4/10-50 μM fluorescent azide (F278701-5 mg, Aladdin, Shanghai, China)/100 mM ascorbic acid (added last from a 0.5 M stock in water). After staining, the cells were washed several times with TBST and then mounted in Antifade Reagent with DAPI (#8961, Cell Signaling Technologies, Danvers, MA, USA) for imaging under a microscope.
Antifade reagent with dapi
Manufactured by Cell Signaling Technology
Sourced in United States
Antifade Reagent with DAPI is a laboratory product designed to preserve fluorescence signals in immunofluorescence or in situ hybridization experiments. It contains an antifade agent that helps maintain the brightness of fluorescent dyes and DAPI, which is a nuclear stain that binds to DNA.
Automatically generated - may contain errors
Lab products found in correlation
Beyoclick edu cell proliferation kit with alexa fluor 488, by Beyotime (1 mentions)
Ab150120, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Alexa fluor 647 conjugate, by Cell Signaling Technology (1 mentions)
Fv1000, by Olympus (1 mentions)
Bz x800 fluorescence microscope, by Keyence (1 mentions)
5 protocols using antifade reagent with dapi
1
Copper-catalyzed Azide-Alkyne Cycloaddition in Gastric Cancer Cells
2
EdU Proliferation Assay in Gastric Cancer
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To conduct the EdU Cell Proliferation Assay, gastric cancer cells were treated with the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (C0071S, Beyotime, Shanghai, China) following the manufacturer's instructions. Briefly, the EdU working solution was added to a 6-well plate, and the cells were cultured for 2 h. The cells were then fixed in a 4 % paraformaldehyde suspension for 20 min and spun onto slides. Subsequently, the cells were permeabilized with 0.5 % PBS-Triton X-100 for 10 min, washed three times, and treated with 0.5 ml of Click reaction solution per well for 30 min at room temperature in the dark. The slides were then washed three times with washing solution and mounted in Antifade Reagent with DAPI (#8961, Cell Signaling Technologies, Danvers, MA, USA) for microscopy analysis.
3
Immunofluorescent Staining Procedure
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To conduct immunofluorescent staining, the cells were fixed in a 4 % paraformaldehyde suspension and spun onto slides. Then, they were permeabilized with 0.5 % PBS-Triton X-100 for 10 min and blocked with 5 % goat serum for 1 h. Subsequently, the slides were incubated overnight with the indicated antibodies at 4 °C, followed by the appropriate secondary antibody, either 488 conjugated goat anti-mouse IgG (ab150120, Abcam, Cambridge, UK) or 594 conjugated goat anti-mouse IgG (ab150077, Abcam, Cambridge, UK). The slides were mounted in an Anti-fade Reagent with DAPI (#8961, Cell Signaling Technologies, Danvers, MA, USA) and analyzed using microscopy.
4
Immunofluorescence Analysis of YAP in PCa Cells
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PCa cells (2 × 104) were cultured on coverslips in a 24-well plate and grown to 70% confluence. The cells were then washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde. After rinsing with PBS, the cells were blocked with PBS containing 1% Triton X-100. Following incubation with anti-YAP rabbit antibody (Alexa Fluor® 647 Conjugate, #38707; Cell Signaling Technology) at 4 °C overnight, the cells were stained with antifade reagent with DAPI (#8961, Cell Signaling Technology) to visualize the nuclei. Finally, the cells were rinsed in PBS and examined using a confocal laser scanning microscope system Olympus FV1000 (Olympus Medical Systems, Tokyo, Japan).
5
PD-L1 Expression Analysis in Cell Lines
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Each cell line was cultured for 48 hours, and the cells were fixed on chamber slides using 4% paraformaldehyde for 10 minutes and permeabilized using 100% ice‐cold methanol for 10 minutes. After blocking the cells with 3% bovine serum albumin for 60 minutes, the slides were stained overnight at 4°C using primary antibodies targeting PD‐L1 (1:200). Next, secondary antibodies targeting rabbit IgG (1:1000) were added for 60 minutes. Staining was performed using the Antifade Reagent with DAPI (Cell Signaling Technology, Danvers, MA), and the cells were observed under a Keyence BZ‐X800 fluorescence microscope (Osaka, Japan).
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