Immunofluorescence was performed as previously described (Sui et al., 2021 (link)). HEK293T or HepG2 cells cultured on 12-mm coverslips were transfected with the indicated plasmids. After 24 h, cells were fixed with 4% paraformaldehyde and permeated with 0.5% Triton X-100. After cells were washed with PBST, they were blocked in 1% BSA and stained with primary antibodies, followed by staining with CoraLite 594 or 488-conjugated IgG secondary antibodies. Nuclei were stained with DAPI (Yesen Biotechnology, Shanghai, China). Fluorescence images were obtained and analyzed using a confocal microscope (FV3000, OLYMPUS).
4 6 diamidino 2 phenylindole (dapi)
Manufactured by Yesen
Sourced in China
DAPI is a fluorescent dye used for staining and visualizing DNA in biological samples. It binds to the AT-rich regions of DNA, allowing the nuclei and chromosomes to be easily identified and analyzed under a fluorescence microscope.
Automatically generated - may contain errors
Lab products found in correlation
Fv3000, by Olympus (4 mentions)
Lsm 880, by Zeiss (4 mentions)
Immunohistochemical blocking solution, by Beyotime (2 mentions)
Ab150160, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab150116, by Abcam (1 mentions)
A1r eclipse ti confocal microscope, by Nikon (1 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ifluor 488 phalloidin, by Yesen (1 mentions)
Tigar, by Abcam (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Alexa 594 conjugated goat anti mouse igg, by Abcam (1 mentions)
Alexa 488 conjugated goat anti rabbit igg, by Abcam (1 mentions)
10 protocols using 4 6 diamidino 2 phenylindole (dapi)
1
Immunofluorescence Imaging of Transfected Cells
2
Immunofluorescence Staining in HEK293T and HepG2 Cells
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HEK293T or HepG2 cells cultured on 12-mm coverslips were transfected with indicated plasmids. After 24 h, cells were fixed with 4% paraformaldehyde, and permeated with 0.5% Triton X-100. After cells were washed with PBST, they were blocked in 1% BSA and stained with primary antibodies, followed by staining with CoraLite 594- or CoraLite 488-conjugated IgG secondary antibodies.48 (link) Nuclei were stained with DAPI (Yesen Biotechnology, Shanghai, China). Fluorescence images were obtained and analyzed using a confocal microscope (FV3000, OLYMPUS).
3
EdU Proliferation Assay in 24-well Plate
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Cells were cultured in a 24-well plate. When they grow to a suitable density, cell transfection was conducted. After stimulation for 48 hours, EdU (30 uM) was added to the culture medium of the 24-well plate and then incubated for 2 hours. The cells were washed with PBS, cell fixation solution was added and incubated at room temperature for 25 min. After washing the cells with PBS, 2 mg/mL glycine was added and incubated on a shaker for 5 min. After washing the cells with PBS, 0.5% Triton X-100 (Ye Sen, Shanghai, China) was added and incubated for 10 min. Apollo staining solution was added and incubated for 30 min in the dark. After washing the cells with PBS, DAPI (Ye Sen, Shanghai, China) was added and incubated for 5 min in the dark. The staining results were observed under a fluorescent inverted microscope, and the results were analyzed with Image J software.
4
Histopathological and Immunofluorescent Analysis of Wound Healing
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On the third and tenth day after the first supernatant treatment, the mice were euthanized via cervical dislocation, and samples were harvested for tissue H&E staining and IF. Briefly, the tissues were fixed with 4% paraformaldehyde for 48 h and then covered with paraffin before sectioning and histological analysis. Blocks were cut into 4 μm thick sections and stained with H&E (YESEN). For IF assay, Briefly, Samples from the wound bed on day 3 were first dewaxed and rehydrated and then repaired antigen via boiling in a 100 °C citrate buffer water bath for 25 min. The tissue sections were blocked with immunohistochemical blocking solution (Beyotime, China) for 90 min. The primary antibodies used in this experiment were incubated at 4 °C overnight, as follows: F4/80 (Rat IgG, monoclonal, Abcam Cat. No.: ab6640), CD86 (Rabbit IgG, Polyclonal, SAB Cat. No.: 32223-2) and CD163 (Mouse IgG, monoclonal, GeneTex Cat. No.: ED2). Then incubated with the following secondary antibodies for 90 min at room temperature: Alexa 594-conjugated goat anti-Rat IgG (ab150160, Abcam), Alexa 488-conjugated goat anti-rabbit IgG (ab150077, Abcam) and Alexa 594-conjugated goat anti-mouse IgG (ab150116, Abcam). The nuclei were stained with DAPI (YESEN, China). Images were acquired using a laser-scanning confocal microscope (Carl Zeiss LSM880, Germany).
5
Immunofluorescence Analysis of Transcription Factors
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After the RAW264.7 cells (1 × 104 cells per well) were seeded in chamber slides for 24 h, the cells were washed twice with ice-cold PBS and then fixed with 4% formaldehyde for 15 min at RT. After washing with PBS for three times, the cells were permeabilized by incubation with 400 μL of a 0.25% Triton X-100 solution for 30 min at 37 °C and followed by incubated for 10 min at room temperature. After blocking the cells with 3% BSA for 1 h, the cells were incubated with the polyclonal antibodies for NF-κB/p65, AP-1/c-Jun, and IRF3 diluted at a 2% BSA solution followed by incubation overnight at 4 °C. After being washed 3 times with PBS, the cells were incubated with the Alexa Fluor 488-conjugated secondary antibody for 1 h at RT. The nuclei were stained with DAPI (YESEN, Shanghai, China), and the fluorescence was visualized using the Nikon A1R Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan).
6
Immunofluorescence and FRET Analysis of Transfected Cells
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HEK293T or HepG2 cells cultured on 12 mm coverslips were transfected with indicated plasmids. For IFA analysis, cells were fixed with 4% paraformaldehyde after 24 h, and permeated with 0.5% Triton X-100. The cells were washed with PBST, blocked in 1% BSA, and stained with primary antibodies, followed by staining with CoraLite 594 or CoraLite 488 conjugated IgG secondary antibodies. Nuclei were stained with DAPI (Yesen Biotechnology, Shanghai, China). Fluorescence images were obtained and analyzed using a confocal microscope (FV3000, OLYMPUS). For FRET analysis, cells were fixed with 4% paraformaldehyde, CFP or YFP images and FRET analysis were performed on Nikon AXR Laser confocal microscope.
7
Nanoparticle-Based Cancer Treatment
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5-FU was purchased from Sangon (Shanghai, China) and DOX was obtained from Meilun Biology (Dalian, China). PLGA in powder form (molecular weight of 5 × 105 Dalton, with a lactide to glycolide ratio of 75:25) was bought from Daigang (Jinan, China) and gelatin was obtained from Dingguo (Beijing, China). Chitosan, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were all obtained from Aladdin (Shanghai, China). Glutaraldehyde, N-N dimethylformamide (DMF), and citric acid were purchased from China Pharmaceutical Group (Beijing, China). Calcein (AM) and DAPI were purchased from Yesen Biotech (Shanghai, China). Hematoxylin and eosin (H&E) solution and the Masson dye kit were obtained from Soledad (Beijing, China).
8
Immunofluorescence Staining of Cochlear Cells
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After culture, cochlea explants or HEI-OC1 cells on coverslips were fixed with 4% paraformaldehyde and blocked for 1 h at room temperature with PBT-1 (5% donkey serum, 0.1% Triton X-100, 1% bovine serum albumin and 0.02% sodium azide in PBS). The samples were then incubated overnight at 4℃ with primary antibodies diluted in blocking solution against cleaved-Caspase 3 (1:500 dilution; Cell Signaling Technology) and TIGAR (1:800 dilution; Abcam). The next day, the samples were incubated with secondary fluorescent antibodies (1:1000 dilution, Invitrogen) along with DAPI (1:1000 dilution, Sigma-Aldrich) in 0.1% Triton X-100 and 1% bovine serum albumin in PBS at room temperature for 1 h. The coverslips were mounted and imaged using a confocal laser scanning microscope (Leica SP8; Leica, Germany).
For the staining of phalloidin, after fixation, the basilar membranes on coverslips were incubated with iFluor™ 488 phalloidin (1:1000 dilution; Yesen) in PBS along with DAPI (1:1000 dilution) for 30 min in the dark. The coverslips were then mounted and confocal microscopy images were acquired.
For the staining of phalloidin, after fixation, the basilar membranes on coverslips were incubated with iFluor™ 488 phalloidin (1:1000 dilution; Yesen) in PBS along with DAPI (1:1000 dilution) for 30 min in the dark. The coverslips were then mounted and confocal microscopy images were acquired.
9
SARS-CoV-2 Nucleocapsid Protein Immunofluorescence
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Caco-2 and HEK293T-hACE2 cells were cultured on coverslips in 24-well plates. After the viral infection and drug treatment for 48 h, as described above, cells were fixed with 4% paraformaldehyde (Biotopped) at dark for 30 min, the cells were then carefully washed, and permeated with 0.5% Triton X-100 (Yeasen Biotech) in PBS for 15 min. Cells were blocked in 1% BSA at room temperature for 1 h, and stained with the monoclonal antibody of anti–SARS-CoV-2 nucleocapsid protein (N protein) (CSB-DA701BmN, Wuhan Huamei Biotech) at 4 °C overnight. The cells were then washed three times with PBS+0.1%BSA, and incubated with CoraLite 594- and CoraLite 488-conjugated IgG secondary antibodies (Proteintech). Nuclei were stained with DAPI (Yesen Biotechnology). Fluorescence images were analyzed with a fluorescence microscope (Nikon) or a confocal microscope (FV3000, Olympus).
10
Immunohistochemical Analysis of Wound Bed
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Samples from the wound bed on day 3 were first dewaxed and rehydrated and then repaired antigen via boiling in a 100 ℃ citrate buffer water bath for 25 min. The tissue sections were blocked with immunohistochemical blocking solution (Beyotime, China) for 90 min. The primary antibodies used in this experiment were incubated at 4 ℃ overnight, as follows: CD86 (Signalway Antibody, USA) and CD163 (GeneTex, USA) and then incubated with the following secondary antibodies for 90 min at room temperature: Alexa 488-conjugated goat anti-rabbit IgG (Abcam, UK) and Alexa 594-conjugated goat anti-mouse IgG (Abcam, UK). The nuclei were stained with DAPI (YESEN, China). Images were acquired using a laser-scanning confocal microscope (Carl Zeiss LSM880, Germany).
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