The cardiac tissues in different groups were prepared into frozen sections for immunofluorescence analysis. The 4.5-um frozen section and cardiac fibroblasts were fixed for 20 min in 4% paraformaldehyde (PFA) at room temperature. After fixation, they were blocked with 5% bovine serum albumin (BSA) in Tris Buffered Saline With Tween (TBST) for 1 h, and then incubated with appropriate primary antibodies at 4 °C over night and incubated with second antibodies (Alexa Fluor ®488-conjugated affinipure goat anti-rabbit lgG (H+L), CyTM 3-conjugated affinipure donkey anti-goat++ lgG (H+L), and Alexa Fluor ®488-conjugated affinipure donkey anti-goat++ lgG (H+L) (Jackson ImmunoResearch Laboratories, Inc., PA, USA)) for 1 hour at room temperature. After that, sections and cells were stained with 1.5 μM 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Sigma, St. Louis, Missouri, USA) for 10 min. The image analysis of frozen sections was performed using a software program (Olympus, Japan). Protein localization of cells was observed and captured with a laser scanning confocal microscope (LSM5, Zeiss, Jena, Thuringia, Germany).
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi
Manufactured by Merck Group
Sourced in United States, China, Germany
2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) is a fluorescent dye commonly used in biological research. It binds to DNA and emits blue fluorescence when excited by ultraviolet light. DAPI is a versatile tool for labeling and visualizing nucleic acids in a variety of applications, including cell and nuclear staining, flow cytometry, and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (18 mentions)
Zen 2012, by Zeiss (9 mentions)
Cyanine3 (cy3), by Beyotime (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Anti fade fluoromount solution, by Beyotime (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Anti fade medium, by Merck Group (2 mentions)
Lsm710 fluorescence microscope, by Zeiss (2 mentions)
Magnesil total rna mini isolation system, by Promega (2 mentions)
N hydroxysuccinimide, by Merck Group (2 mentions)
Cy3 labeled goat anti rat igg, by Beyotime (2 mentions)
3 3 dioctadecyloxacarbocyanine dioc18 3, by Beyotime (2 mentions)
Tcs sp5, by Leica (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Jc 1 mitochondrial membrane potential detection kit, by Keygen Biotech (2 mentions)
Software program, by Olympus (1 mentions)
52 protocols using 2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi
1
Immunofluorescence Analysis of Cardiac Tissues
2
Cytoskeletal Actin and Cell Nuclei Analysis
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AZ31 alloy and coating samples were placed in 24-well culture plates. Then 1.5-ml cell suspension with a density of 7.0 × 104 cell/ml was seed on the specimens. After incubation for 1, 4 and 24 h, the cells were gently rinsed with PBS three times. Then the cells were fixed, permeabilized and blocked with 4% paraformaldehyde solution, 0.1% (v/v) Triton X-100 and 1 wt% BSA solution, respectively. Subsequently, the cells were stained with Fluorescein Isothiocyanate (FITC)-Phalloidin and further staining with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Sigma, USA). The cytoskeletal actin and cell nuclei were characterized with a fluorescence microscopy (Olympus, Japan).
3
Investigating Cell Adhesion on Biomaterials
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For cell adhesion, the rBMS cells were cultured on the sample surface for 1 day and 3 days. At the end of each time point, the culture medium was removed, and the samples were rinsed thrice with PBS and then immobilized with 4% paraformaldehyde solution overnight. Subsequently, the fixed cells were incubated using fluorescein isothiocyanate-Phalloidin (FITC-Phalloidin, Yeasen Biotech, China) for 1 h and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Sigma, USA) for 10 min in darkness. Cell morphology was visualized using confocal laser-scanning microscopy (CLSM, from Nikon A1R, Japan). The cell morphology of rBMS cells on SPK, SNP, TAP and STP was observed using SEM after dehydrated in ascending concentrations of ethanol solutions (30%, 50%, 75%, 90%, 95% and 100%, v/v) for 10 min and drying at 25 °C.
4
Cytotoxicity Evaluation of Aspirin and 5-FU
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The analyzed compounds (aspirin and 5-fluorouracil) and the other reagents used in the present study dimethyl sulfoxide (DMSO), fetal calf serum (FCS), penicillin/streptomycin, trypsin-EDTA solution, phosphate saline buffer (PBS), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 4′,6-Diamidino-2-phenylindole dihydrochloride, and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), were purchased from Sigma Aldrich, Merck KgaA (Darmstadt, Germany), and Alexa Fluor® 555 Phalloidin was acquired from Cell Signaling USA.
Cell lines were cultured in the specific media DMEM (P04-03550) and McCoy’s 5A (P04-05500), which were purchased from PAN Biotech GmbH (Aidenbach, Germany). As part of RT-PCR, the following primers were used: 18S, Bax, Bcl-2 purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA) and Bad, caspase 3, and caspase 8, purchased from Eurogentec (Seraing, Belgium). All reagents presented appropriate characteristics for use in cell culture.
Cell lines were cultured in the specific media DMEM (P04-03550) and McCoy’s 5A (P04-05500), which were purchased from PAN Biotech GmbH (Aidenbach, Germany). As part of RT-PCR, the following primers were used: 18S, Bax, Bcl-2 purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA) and Bad, caspase 3, and caspase 8, purchased from Eurogentec (Seraing, Belgium). All reagents presented appropriate characteristics for use in cell culture.
5
Biocompatible Silkworm Cocoon-Based Materials
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BC bulks were obtained from Hai Nan Yeguo Foods Co., Ltd. (Haikou, China). Silkworm cocoons from B. mori were purchased from Tongxiang City, Zhejiang Province, China. Horseradish peroxide was purchased from Sigma Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (USA). Calcein acetoxymethylester/ Propidium iodide (Calcein AM/PI) were purchased from Yeasen Biotech Co., Ltd. (Shanghai, China). Rhodamine phalloidin, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (USA). Rabbit anti-Ki67, rabbit anti-p63 and fluorescein isothiocyanate (FITC) were purchased from Abcam (UK). All other chemicals were acquired from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), were analytical grade and used as received.
6
Immunofluorescence Assay for rHcES-24 Binding
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Freshly isolated PBMCs were incubated in the presence and absence (control) of rHcES-24 (5μg/ml) for 1 h at 37°C. Confirmation of binding was determined by an immunofluorescence assay (IFA) as described by Yuan et al. [54 (link)]. Briefly, washed cells (105 / ml) were fixed with 4% paraformaldehyde on a poly-L-lysine-coated glass slide. The cells were then treated with blocking solution (4% BSA in PBS) for 30 min to minimize background staining. After sequential incubation with rat anti-rHcES-24 IgG (1:100) for 2 h and a secondary antibody (1:300) coupled to the fluorescent dye Cy3 (Beyotime, Jiangsu, China) for 1 h, nuclear staining with 2-(4-amidinophenyl)-6-indole carbamidinedihydrochloride (DAPI, 1.5 μM; Sigma, MO, USA) was performed for 6 min. Then, protein localization was determined by observing the staining patterns with a 100× oil objective lens on a laser scanning confocal microscope (L SM710, Zeiss, Jena, Germany). Digital images were captured using the Zeiss microscope software package ZEN 2012 (Zeiss, Jena, Germany).
7
Tracking Transplanted hPD-MSCs in Ovaries
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To track and locate the transplanted hPD-MSCs in the ovarian tissues, the cells were prelabeled with PKH67 Green Fluorescent Cell Linker kits (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, a total of 2 × 107 hPD-MSCs were washed and gently resuspended in 1 ml of Diluent C. In parallel, 4 μl of PKH67 dye was added to 1 ml of Diluent C (4 × 10−6 M) and incubated with the hPD-MSC solution for 5 min. To bind excess dye, the same volume of 1% BSA was added. The labeled hPD-MSCs were washed and observed by fluorescence microscopy.
PKH67-labeled hPD-MSCs were transplanted into rats via the tail vein. Ovaries were fixed with OCT compound and made into fresh sections (12 μm thick). After fixation with 4% paraformaldehyde for 20 min, the sections were washed and incubated with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Sigma-Aldrich) at room temperature for 10 min. The sections were then imaged under a laser scanning confocal microscope (LSCM; Leica, Wetzlar, Germany).
PKH67-labeled hPD-MSCs were transplanted into rats via the tail vein. Ovaries were fixed with OCT compound and made into fresh sections (12 μm thick). After fixation with 4% paraformaldehyde for 20 min, the sections were washed and incubated with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Sigma-Aldrich) at room temperature for 10 min. The sections were then imaged under a laser scanning confocal microscope (LSCM; Leica, Wetzlar, Germany).
8
Immunocytochemistry of Cell Signaling
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A total of 5 × 104 Cells was seeded in the confocal dish. After 2 days of culture, the cells were washed with phosphate buffer saline (PBS), fixed in 4% paraformaldehyde, and cell membranes were permeabilized with 0.3% Triton X-100 solution. Next, cells were blocked with 5% bovine serum albumin (BSA) in 1% Triton X-100 and then blotted at 4°C overnight with different primary antibodies against LEF1 (1:200 CST), β-catenin (1:200 Abcam). Fluorescence-conjugated secondary antibodies (Alexa Fluor antibody, Life Technologies) were then added to these dishes. Cells were further washed and the nuclei were stained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Sigma). These confocal dishes were visualized using a Carl Zeiss confocal fluorescence microscope (LSM 780).
9
Immunofluorescence Staining for BDNF and GFAP
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Specimens were pre-treated for 5 minutes in 0.01 M citrate buffer, pH = 6.0 at 90 °C, autofluorescence was quenched by 15 min incubation in 0.1 M Glycine-tris-buffered saline (TBS) at room temperature (RT), then unspecific binding was blocked with 10% Normal Goat Serum/0.5% Triton-TBS solution for 30 min at RT. Primary antibodies TBS-dilutions were applied overnight at +4 °C. As primary antibodies, we used rabbit anti-BDNF (Santa Cruz, sc-546, dilution 1:100) and mouse anti-GFAP (Sigma, G3893, dilution 1:500). Species-matching secondary antibodies (AlexaFluor 488 or 594, Thermo, dilution 1:500) were applied for 3 hours at RT. Nuclei were counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Sigma). Samples were evaluated using the Zeiss Axio Scope.A1 fluorescent microscope (Zeiss, Oberkochen, Germany).
10
Immunofluorescence Staining of rHcABHD
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Freshly collected female and male adults (20 ~ 30 for each) were washed, dehydrated, fixed, embedded and cut into cryostat sections as previous described (22 (link)). To block non-specific binding, the cryosections were treated with 10% normal goat serum in PBS containing 0.1% Tween-20 (PBST) at room temperature for 1 h. The sections were then incubated with rat anti-rHcABHD IgG (1:200) or normal rat IgG (control) at 4°C overnight. After three washes in PBST, the sections were then incubated with Cy3-labeled goat anti-rat IgG (1:500) for 1 h at 37°C. Following three washes in PBST, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Sigma-Aldrich) were used for DNA staining at room temperature for 5 min. The samples were then immersed into anti-fade medium (Sigma-Aldrich) to prevent fluorescence fading for microscopic examination. Finally, the sections were imaged using LSM710 fluorescence microscope (Zeiss, Jena, Germany) at 60× magnification and digital images were analyzed using ZEN 2012 software (Zeiss).
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