HepG2 cells were inoculated into 12-well plates containing sterile cell climbing tablets and then cultured in an incubator with SBP-2A for 48 h. According to the instructions provided with the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Beyotime, C0078S), an equal volume of EdU solution (20 μM) was added, and the cells were incubated for 2 h, followed by fixing with 4% paraformaldehyde. Then, 0.3% Triton X-100 was added at 200 μl/well for cell permeabilization (Solarbio, T8200), and the cells were placed in 1 × Apollo 567 reaction solution at room temperature for 30 min. Then, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Beyotime, C1005) was used to counterstain the cells for 10 min. Finally, the images were captured by the DMI 6000B Leica microsystem in a live cell imager (Wetzlar, Germany), and the percentage of EdU-positive cells in each group and each well was randomly counted in five fields of view.
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi
Manufactured by Beyotime
Sourced in China
2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) is a fluorescent dye commonly used in molecular biology and cell biology applications. It is a DNA-binding agent that exhibits high affinity for adenine-thymine (A-T) rich regions of the DNA molecule. DAPI emits blue fluorescence when bound to DNA, allowing visualization and detection of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
1 1 dioctadecyl 3 3 3 3 tetramethylindocarbocyanine perchlorate dii, by Beyotime (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
3 3 dioctadecyloxacarbocyanine perchlorate dio, by Beyotime (3 mentions)
Eu5888, by Leica (3 mentions)
Anti wdr79, by Fortis Life Sciences (3 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (3 mentions)
Dspe peg2000 fa, by Xi’an Ruixi (3 mentions)
Immunostaining blocking buffer, by Beyotime (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Eclipse ni fluorescent microscope, by Nikon (2 mentions)
Triton x 100, by Solarbio (2 mentions)
Bovine serum albumin (bsa), by Solarbio (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (2 mentions)
Alexa fluor 488 labeled secondary antibody, by Beyotime (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
2 7 dichlorofluorescin diacetate dcfh da, by Beyotime (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Cell counting kit 8 cck 8, by Beyotime (2 mentions)
Anti runx2, by Abcam (2 mentions)
Anti col1a1, by Abcam (2 mentions)
75 protocols using 2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi
1
Evaluating Cell Proliferation in HepG2 Cells
2
Immunostaining of Cardiac Ion Channels
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Cells were fixed in 4% paraformaldehyde (Boster) for 30 min, and washed in PBS (10 min × 3 times). To block non-specific epitopes, cells were incubated with immunostaining blocking buffer (Beyotime Institute of Biotechnology) for 60 min. Then cells were incubated overnight at 4°C with primary antibodies: c-kit (sc-1494, 1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), HCN1 (ab84816, 1:100), HCN2 (ab65704, 1:100), HCN3 (ab84818, 1:100) and HCN4 (ab69054, 1:100) (all from Abcam), followed by the appropriate fluorescence-conjugated secondary antibodies: Alexa Fluor 488 mouse anti-goat IgG (bs-0294M, 1:200; Bioss, Beijing, China), Alexa Fluor 647 goat anti-mouse IgG (P0191, 1:200) and Alexa Fluor 647 goat anti-rabbit IgG (P0180, 1:200) (both from Beyotime Institute of Biotechnology). Next, cells were incubated with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime Institute of Biotechnology) to label the cell nucleus. Negative control was performed by omitting the primary antibody. All incubation steps were followed by washes with PBS (10 min × 3 times). Cells were visualized and photographed using a confocal laser scanning microscope (Leica, Wetzlar, Germany). The mean fluorescence density was measured by Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
3
DOTAP-based Nanolipoplex Characterization
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1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from Cardenpharma Switzerland LLC (Liestal, Switzerland). Cholesterol, Ham’s F12 nutrient medium, high glucose DMEM, fetal bovine serum (FBS), and lipopolysaccharide from Escherichia coli 055:B5 were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Maleimide derivatized PEG2000-DSPE (Mal-PEG2000-DSPE) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). TAT peptide with a terminated cysteine (Cys-TAT, AYGRKKRRQRRR; MW: 1734) was synthesized by Guoping Pharmaceutical Corporation (Hefei, Anhui, China). DHA was obtained from the ZheJiang Institute for Food and Drug Control (Hangzhou, Zhejiang Province, China). Anti-HMGB1, -MyD88, and -NF-кB antibodies were purchased from Abcam (Cambridge, UK). Anti-TLR4 and -IRAK4 antibodies were purchased from Cell Signaling Technology (Beverly, MA). 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was purchased from Beyotime Biotechnology (Hangzhou, Zhejiang, China). Cy3-labeled NC siRNA and negative control siRNA (NC) were synthesized by GenePharma (Shanghai, China). HMBG1 siRNA and TRIzol reagent were purchased from Thermo Fisher (Waltham, Massachusetts, USA). HEK-Blue Selection and QUANTI-Blue solution were purchased from Invivogen (San Diego, CA, USA).
4
Nrf2 Immunostaining in Tissue Sections
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Immunohistochemistry and immunofluorescent staining were performed as previously described [52 (link)]. In detail, the tissue sections (4 mm) were incubated with Nrf2 antibody, (Sigma, St. Louis, MO, USA), which was diluted in Antibody Diluent Reagent Solution (Invitrogen, Carlsbad, CA, USA) and reactions were carried out overnight at 4 °C. After washing with PBS for three times (5 min each time), the sections were incubated with horse anti-mouse antibody (Sigma, Louis, MO, USA) at room temperature for 30 min. After washing three times with PBS, the slides were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime Biotech Inc., Nantong, China) for 2 min to show the locations of nuclei. Images were acquired using an Olympus BX40 microscope (Olympus Corporation, Tokyo, Japan). The Image-pro Plus 5.0 (Media Cybernetics, Inc., Rockville, MD, USA) was applied to calculate the expression of Nrf2.
5
Immunostaining of Brain Slices for NRF2
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The brain slices were rinsed three times with PBS for 5 min each and blocked in 5% BSA for 1 h. The primary antibody (rabbit anti-NRF2 antibody, 1:500,16396–1-AP, Proteintech) was incubated with the slices overnight at 4 °C. The next day, the slices were rewarmed in the primary antibody for 1 h at 24±1 °C and then washed three times for 5 min each time with 0.3% PBST. The secondary antibody (goat anti-rabbit IgG, 1:500, SA00013–4, Proteintech) was incubated at room temperature for 1 h in a wet box. Then, slices were washed in 0.3% PBST three times for 5 min each time and stained with 2–4-Amidinophenyl-6-indolecarbamidine dihydrochloride (DAPI, P0131, Beyotime) for 5 min. Fluorescence was captured on a LEICA DMI8 inverted microscope system.
6
Immunofluorescence Staining Assay for Ki67
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Cells were mounted onto microscope slides with a 30-min fixation using 4% formaldehyde, followed by 10-min permeabilization using 0.5% Triton X-100 (Solarbio, China). After 30-min blocking using 1% bovine serum albumin (Solarbio, China), the cells were incubated with antibody ab15580 (anti-Ki67, Abcam) overnight at 4°C; thereafter, they were further incubated in the dark with a secondary antibody (Beyotime Biotechnology, China) for 30 min at 37°C. For the cell nuclei staining, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) from Beyotime Biotechnology was used. The ECLIPSE Ni Fluorescent microscope (Nikon) was used for visualization.
7
Immunofluorescence Imaging of Mouse Brain Tissues
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Mouse brain tissues fixed in 4% paraformaldehyde (PFA) for 24 h were then dehydrated with 30% sucrose for 48 h and sectioned using a cryostat into 5 μm sections. Next, the sections were rewarmed for 30 min, fixed in acetone for 10 min, washed with PBS three times, permeabilized with 0.3% Triton X-100 for 10 min, incubated with a blocking solution (3% bovine serum albumin in PBS) for 1 h and then incubated with indicated antibodies at 4 °C overnight. On the following day, sections were washed three times with PBS (5 min each time), incubated with corresponding secondary antibodies coupled with Alexa Fluor® 488 or Alexa Fluor® 594 at room temperature for 1 h under dark conditions, then washed and mounted with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Beyotime, Wuhan, China). Images of fluorescence were captured with an LSM880 confocal laser-scanning inverted microscope (ZEISS, Jena, German).
8
Targeted Delivery of PLGA Nanoparticles to Caco-2 Tumors
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A total of 1×107 Caco-2 cells were implanted subcutaneously into the right flank of BALB/c mice. The PLGA NPs were fluorescence labeled with green DiO dye and divided into four equal groups. The four groups acted as PLGA NPs (P group), RBCm–PLGA NPs (RP group), RBCm–PLGA NPs with free anti-EGFR-iRGD proteins (iE + RP group), and RBCm–PLGA NPs modified with anti-EGFR-iRGD proteins (iE–RP group). The samples were injected to Caco-2 tumor-bearing nude mice via the tail vein. Tumor-bearing nude mice were sacrificed after 24 h, and subcutaneous tumors were collected. Immunofluorescence analysis was carried out with frozen tumor tissues. For immunostaining, slides were first blocked with 3% bull serum albumin (BSA) for 1 h at room temperature and then exposed to the primary antibody against CD31 (1:300 dilution; GeneTech, Shanghai, People’s Republic of China) at 4°C overnight and the second antibody (1:300 dilution) at 37°C for 30 min. Finally, nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime) and slides were visualized using CLSM.
9
Oridonin-induced Apoptosis and Oxidative Stress
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Oridonin (≥98%, HPLC) is obtained from Mingwang biotechology (China). Fetal calf serum (FCS), penicillin/streptomycin, dulbecco’s modified eagle medium (DMEM), and trypsin kit are obtained from Gibco (USA). Paraformaldehyde is purchased from Sigma (USA). 3-(4, 5)-dimethylthiazo(-z-y1)-3,5-diphenyt- etrazoliumromide (MTT), N-acetyl-L-cysteine (NAC), Annexin V-FITC/PI (Annexin V-Fluorescein Isothiocyanate/Propidium Iodide) apoptosis detection kit, DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) reactive oxygen species assay kit, rhodamin 123, actin-tracker green (phalloidin- Fluorescein Isothiocyanate), 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and cell cycle analysis kits (Propidium Iodide) are purchased from Beyotime Institute of Biotechnology, China.
10
Doxorubicin-Loaded Mesoporous Silica Nanoparticles
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Zinc acetate, calcium chloride anhydrous (CaCl2), lithium hydroxide (LiOH) and N,N-dimethyl formamide (DMF) were purchased from Xilong Chemical Industry (Shantou, China). 3-aminopropyltriethoxysilane (APTES), sodium phosphate dibasic dodecahydrate (Na2HPO4∙12H2O) and hexane were purchased from Aladdin (Shanghai, China). Cetyltrimethylammonium bromide (CTAB) was purchased from Thermo Fisher Scientific (Shanghai, China). Tetraethylorthosilicate (TEOS), ammonia (NH3∙H2O), absolute ethanol, succinic anhydride and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sinopharm (Beijing, China).
Doxorubicin hydrochloride (DOX) was purchased from Adamas-beta (Shanghai, China). Dulbecco’s modified eagle medium (DMEM) was purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang biological technology (Hu Zhou, China). 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and penicillin-streptomycin solution were purchased from Beyotime (Shanghai, China). A cell counting kit-8 (CCK-8) was purchased from DOJINDO Laboratories (Kumamoto, Japan). The prolong antifade kit was purchased from Life Technologies (Eugene, OR, USA) and 4% paraformaldehyde was purchased from Vicmed (Xuzhou, China).
Doxorubicin hydrochloride (DOX) was purchased from Adamas-beta (Shanghai, China). Dulbecco’s modified eagle medium (DMEM) was purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang biological technology (Hu Zhou, China). 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and penicillin-streptomycin solution were purchased from Beyotime (Shanghai, China). A cell counting kit-8 (CCK-8) was purchased from DOJINDO Laboratories (Kumamoto, Japan). The prolong antifade kit was purchased from Life Technologies (Eugene, OR, USA) and 4% paraformaldehyde was purchased from Vicmed (Xuzhou, China).
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