Cells seeded into a 24-well plate were starved in the serum-free medium for 4 h before the treatment with a TGR5 agonist (lithocholic acid) for 72 h. Following treatment, at room temperature, we fixed the cells with 4% paraformaldehyde for 15 min and permeabilized them with 0.5% Triton X-100 for another 5 min. The actin filaments in cells were characterized after staining with rhodamine phalloidin (Invitrogen, Carlsbad, CA, USA) for 20 min. Then, the nucleus was identified with 4–6-diamidino-2-phenylindole dihydrochloride (DAPI) (Abcam, Cambridge, MA, USA) after staining for 15 min. Cells were imaged under fluorescence microscope (IX71; Olympus, Tokyo, Japan; magnification, ×200) with an imaging system (DP2-BSW) from same supplier. We measured the myocytes that completely observed in the field. Changes in cell size were then quantified using NIH ImageJ software42 .
4 6 diamidino 2 phenylindole dihydrochloride dapi
Manufactured by Abcam
Sourced in United States
4,6-diamidino-2-phenylindole dihydrochloride (DAPI) is a fluorescent dye that binds to DNA. It is commonly used in fluorescence microscopy techniques to visualize and stain cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Dp2 bsw, by Olympus (1 mentions)
Mounting medium, by Abcam (1 mentions)
Sp8 confocal microscopy, by Leica (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg secondary antibody, by Abcam (1 mentions)
Tunel assay, by Beyotime (1 mentions)
Guinea pig anti insulin, by Abcam (1 mentions)
Streptavidin alexa fluor 488, by Abcam (1 mentions)
Lsm 510 confocal laser scanning microscope, by Zeiss (1 mentions)
5 protocols using 4 6 diamidino 2 phenylindole dihydrochloride dapi
1
Characterizing TGR5 Agonist-Induced Actin Cytoskeleton
2
Multiparametric Immunofluorescence Imaging
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Cells were fixed using 4% paraformaldehyde in cold DPBS and permeabilized using 10.5% Triton X-100 in DPBS for three minutes. A blocking solution with 5% bovine serum albumin (BSA) was then added. The cells were incubated with IL-6, FLG, IL-4, and IL-1β primary antibodies overnight, followed by incubation with a FITC-labeled secondary goat anti-Rabbit IgG (H&L) antibody (Abcam) for one hour. Nuclei were stained with 4′,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) included in the mounting medium (Abcam). After, all cells were visualized using a Leica SP8 confocal microscopy (Philadelphia. PA, USA).
3
Immunofluorescence Verification of Neutralization
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Immunofluorescence was used to further verify the neutralization test. Vero cells in plate were washed with phosphate-buffered saline (PBS), fixed in 80% precooled acetone (Sigma-Aldrich, USA) for 30 min, then washed and blocked in 1% bovine serum albumin at room temperature (19-21°C) for 30 min, and then incubated with anti-Spike RBD Rabbit monoclonal antibodies (mAbs) (1:1000; Sino Biological) at 4°C overnight. The plates were washed and added with Alexa Fluor488®-conjugated Goat Anti-rabbit IgG secondary antibody (1:1500; Abcam, Cambridge, UK) at room temperature for 2 hours. 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (2 µM; Abcam) was used to stain the nuclei (Figure S1
5
Islet Morphology Analysis by Immunofluorescence
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The morphology of islets was evaluated by histological analysis and microscopy. Following fixation in 4% (w/v) paraformaldehyde and permeabilization by 0.3% Triton for 3 h, human islets were immunofluorescent labeled for glucagon and insulin. 16 The islets were blocked with 10% normal goat serum/0.15% Triton-X 100/10 mM PBS overnight at 4°C. The primary and secondary antibodies were diluted in 1% BSA/0.2% Triton X-100/10 mM PBS. Staining for glucagon was performed using primary rabbit anti-glucagon (1:100; Abcam, The Netherlands), and two different secondary antibodies including biotin anti-rabbit (1:200; Abcam) and streptavidin-Alexa Fluor 488 (1:200; Abcam) to distinguish alpha from b-cell. In case of insulinpositive b-cells, guinea pig anti-insulin (1:200; Abcam) was used as the primary antibody, whereas Alexa Fluor 647 goat anti-guinea pig (1:500; Abcam) was applied as the secondary conjugated antibody. Nuclei were stained using 4¢,6¢diamidino-2-phenylindole dihydrochloride (DAPI) (Abcam) for 10 min. Each step was followed by subsequent washing in 1% BSA/0.2% Triton X-100 in 10 mM PBS. Samples were subjected to optical sectioning in axial (z) dimension using a Zeiss LSM510 confocal laser scanning microscope.
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