The PK-15 cells were transfected with swine IFITM expression constructs in glass-bottomed coverslips (NEST Biotechnology, Wuxi, China) for 24 h. Then, the cells were fixed with 4% (vol/vol) paraformaldehyde at 4°C for 30 min. Following cell fixation, QuickBlock Blocking Buffer for Immunol Staining (Beyotime, Shanghai, China) was used. All the samples were stained with ANTI-FLAG monoclonal antibody (Sigma-Aldrich) to detect IFITM proteins and anti-LAMP1 monoclonal antibody (Abcam; ab25245) to detect endosomes, and they were then incubated with Alexa Fluor 488 conjugated goat anti-mouse polyclonal antibody (Abcam; ab150113) and Alexa Fluor 594 conjugated goat anti-rabbit polyclonal antibody (Abcam; ab150080). The nuclear DNA was labeled with 4′,6-diamidino-2-phenylindole solution (Beyotime, Shanghai, China). Finally, all the cells were visualized with a Leica DM-IRE2 confocal microscope using a 63× immersion oil objective. Images were captured with the Leica Application Suite advanced fluorescence software (LAS X) and the ImageJ package.
4 6 diamidino 2 phenylindole solution
Manufactured by Beyotime
Sourced in China
4',6-diamidino-2-phenylindole (DAPI) is a fluorescent dye commonly used in molecular biology to stain and identify cell nuclei. It binds strongly to DNA and emits blue fluorescence when excited by ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
Dm ire2 confocal microscope, by Leica (1 mentions)
Glass bottomed coverslips, by NEST Biotechnology (1 mentions)
Alexa fluor 488 conjugated goat anti mouse polyclonal antibody, by Abcam (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit polyclonal antibody, by Abcam (1 mentions)
Quickblock blocking buffer for immunol staining, by Beyotime (1 mentions)
Anti flag monoclonal antibody, by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Beyoclick edu 594 kit, by Beyotime (1 mentions)
Brightred apoptosis detection kit, by Vazyme (1 mentions)
Eclipse c1, by Nikon (1 mentions)
4 protocols using 4 6 diamidino 2 phenylindole solution
1
IFITM Protein Localization in Endosomes
2
Evaluating Cardiac Fibrosis and Antioxidant Stress in Rats
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The rats were euthanized at a determined time after surgery, and hearts with surrounding adhesive tissues were harvested. To evaluate the fibrous tissues, the hearts were fixed with 4% paraformaldehyde for 24 hours and then embedded in paraffin and sliced into 8 μm thick sections along the short axis transversely across the fibrous zone. To evaluate the antioxidative stress, the hearts were frozen, sectioned into 20 μm thick sections. The heart sections for H&E staining were used to evaluate the parameters of adhesion. The adhesion area was evaluated by ImageJ software. For immunofluorescence staining, the heart sections were incubated with anti–MSR-1 rabbit monoclonal antibody (1:300; Bioss, China) or anti–Gata binding factor 6 (GATA6) rabbit monoclonal antibody (1:300; Affnity) and subjected to incubation with goat anti-rabbit Alexa Fluor Cy3-conjugated secondary antibody (1:500; Abcam). Last, the nucleus was stained with 4′,6-diamidino-2-phenylindole solution (Beyotime) and then observed under a fluorescence microscope (Olympus).
3
DNA Synthesis Quantification in Colorectal Cancer Cells
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CRC cells were subjected to an assessment of DNA synthesis using the BeyoClick™ EdU-594 Kit(Beyotime). The cells were plated in a 12-well plate at a density of 2 × 10 5 cells per well. HCT-116 cells received treatment with 5-FU (14 µg/mL), NaB (6 mM), or a combination of both, while SW-480 cells were exposed to 5-FU (8 µg/mL), NaB (7 mM), or their combination for an additional 24 h. After a 2-h incubation with EdU, cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (Biosharp) for 15 min. Following permeabilization with 0.1% Triton X-100 for 15 min, the cells were incubated with a fluorescent marker solution for 30 min. Nuclear staining was performed using 4',6-diamidino-2-phenylindole solution (Beyotime). Subsequently, the cells were sealed and examined under a fluorescence microscope (BX53, Olympus).
4
Quantification of Jejunal Apoptosis
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The apoptotic index of the jejunum was determined using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) BrightRed Apoptosis Detection Kit (#A113; Vazyme Biotech, Nanjing, Jiangsu, China) in accordance with the manufacturer’s guidance. After deparaffinization and aquation, the jejunal specimens were dipped into proteinase K (20 μg/mL) for 20 min at room temperature, followed by incubation with the TUNEL mixture (deoxynucleotidyl transferase enzyme and BrightRed Labeling Mix) at 37 °C for 1 h in a dark and humidified chamber. Subsequently, the slices were counterstained with the 4,6-diamidino-2-phenylindole solution (#P0131; Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) and the images were captured using a fluorescence microscope (Nikon Eclipse C1; Nikon, Tokyo, Japan). The percentage of TUNEL-positive cells in the jejunum was quantified from 15 well-oriented villi each section by an independent and blinded observer who was not aware of the treatment procedures, using Image Pro Plus 6.0 software.
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