After 48 h of co-culture, DAPI staining was carried out as described by Chazotte [26 (link)]. HUVECs-NC, HUVECs-siGRB7-1, or HUVECs-siGRB7-2 on glass slides were washed three times with phosphate-buffered saline (PBS). Cells were fixed with 3.7% formaldehyde for 10 min and rinsed three times with PBS. Next, the cells were treated with 0.2% Triton X-100 for 5 min to permeabilize the cells. After rinsing three times with PBS, cells were incubated with DAPI staining solution (Boster, Wuhan, China) for 10 min. After washing three times with PBS, the cells were covered with antifade mounting medium (Beyotime, Shanghai, China) and observed using a fluorescence microscope.
Dapi staining solution
Manufactured by Boster Bio
Sourced in China, United States
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye that binds to the minor groove of DNA. It is commonly used in microscopy and flow cytometry applications to stain and visualize nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Inverted optical microscope, by Nikon (2 mentions)
Immunol staining wash buffer, by Beyotime (2 mentions)
Immunol staining primary antibody dilution buffer, by Beyotime (2 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Sp8 confocal microscope, by Leica camera (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
Trypsin, by Boster Bio (1 mentions)
Anti mmp13, by Abcam (1 mentions)
Anti p65, by Proteintech (1 mentions)
Anti stat3, by Proteintech (1 mentions)
Anti iκb, by Proteintech (1 mentions)
Collagenase type 2, by Boster Bio (1 mentions)
Alantolactone, by MedChemExpress (1 mentions)
Anti mmp1, by Proteintech (1 mentions)
Anti mmp 3, by Proteintech (1 mentions)
Anti atg5, by Proteintech (1 mentions)
Anti p62, by Proteintech (1 mentions)
Anti inos, by Abcam (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Fitc conjugated affinipure goat anti rabbit igg, by Boster Bio (1 mentions)
11 protocols using dapi staining solution
1
DAPI Staining of HUVEC Cells
2
Apoptosis Quantification in Cardiomyocytes
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The samples were treated with 4% paraformaldehyde for 20 min and blocked with 1% BSA and 0.2% Triton-X for 5 min. Then, they were treated with TUNEL mixture test solution (Beyotime, Shanghai, China) for 1 h at 37 °C, followed by treatment with DAPI staining solution (Boster, Wuhan, China) for 20 min at room temperature. Finally, the results were captured using a fluorescence microscope. The apoptosis index was expressed as the proportion of TUNEL-positive cells to total cardiomyocytes.
3
Histological and Immunofluorescence Analysis of Mouse Cardiac Tissue
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Heart tissues of mice were fixed in 10% phosphate-buffered formalin for 24 hours, dehydrated by ethanol and xylene, and then embedded in paraffin. Cardiac tissue sections were stained with H&E to observe morphological changes and stained with Sirius red to detect collagen deposition. Lipid deposits were observed by oil red staining of frozen sections of heart tissues.
Cardiac tissue sections were stained with primary antibodies of SLMAP (Abcam, USA) and GLP-1R (Affinity, China) overnight at 4°C. After thorough washing, sections were incubated with FITC-labelled anti-rabbit IgG secondary antibodies at 37°C for 50 min. Then, the stained sections were counterstained with DAPI-staining-solution (Boster, USA). Finally, the images were captured under an inverted optical microscope (Nikon, Japan).
Cardiac tissue sections were stained with primary antibodies of SLMAP (Abcam, USA) and GLP-1R (Affinity, China) overnight at 4°C. After thorough washing, sections were incubated with FITC-labelled anti-rabbit IgG secondary antibodies at 37°C for 50 min. Then, the stained sections were counterstained with DAPI-staining-solution (Boster, USA). Finally, the images were captured under an inverted optical microscope (Nikon, Japan).
4
Subcellular Localization of Zebrafish Prkcaa and Prkcab
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Cell culture, transfection and microphotography were performed as previously described [50 (link)]. Briefly, the 293T cells (ATCC number CRL-3216) were cultured in DMEM/High glucose (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (PAN-Biotech, Aidenbach, Germany). The cells were maintained at 37 °C in an incubator (Thermo Fisher, Waltham, MA, USA) supplied with 5% CO2. The plasmids of pAC-Prkcaa-GFP and pAC-Prkcab-GFP were transfected into the 293T cells to characterize subcellular localization of zebrafish Prkcaa and Prkcab. The cells were seeded onto 35 mm glass bottom petri dishes at a density of 2 × 105 cells per dish 1 day before transfection. Additionally, 1 h before transfection, the culture medium was replaced with fresh culture medium. The cells were transfected with 1 μg plasmid and 1 μL VigoFect reagent (Vigorous Biotech, Beijing, China). At 6 h after transfection, the old medium was replaced. At 24 h after transfection, the cells were washed twice with PBS and fixed with 4% PFA. After 3 washes with PBS, the cells were stained with DAPI-staining solution (BOSTER, Wuhan, China). Microphotography was conducted using a SP8 confocal microscope (Leica, Wetzlar, Germany) under a 63× oil immersion objective.
5
Alantolactone Modulates Inflammatory Response
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Alantolactone (HY-N0038) was obtained from MedChem Express (MCE). Recombinant mouse IL-1β cytokine was provided from R&D Systems (501-RL-010, United States). Solarbio supplied safranin O liquor (Beijing, China). Primary antibodies applied in this research were: anti-iNOS, anti-MMP-13 which were acquired from Abcam (Shanghai, China), anti-COX2, anti-LC3Ⅱ/Ⅰ, anti-P-STAT3, anti-P-P65, anti-P-IκB, anti-P/T-mTOR which were purchased from CST (Beverly, MA, United States), anti-STAT3, anti-MMP-1, anti-MMP-3, anti-ATG5, anti-P62, anti-P65, anti-IκB, anti-P/T-PI3K, anti-P/T-AKT, anti-GAPDH which were afforded from Proteintech Group (Wuhan, Hubei, China) and anti-ADAMTS5 which was supplied from Boster Biological Technology (Wuhan, Hubei, China). Secondary antibodies, Cy3 and FITC Conjugated AffiniPure Goat Anti-Rabbit IgG, DAPI staining solution, collagenase type II and trypsin were got from Boster Biological Technology (Wuhan, Hubei, China). HanBio Inc. (Shanghai, China) served the mRFP-GFP-LC3 adenoviral vectors.
6
Phage-Cell Binding Assay Protocol
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SKOV3, Hela, AN3CA, MCF7, MHCC97-H and HEK293 cells (4 × 104/well) were cultured on coverslips overnight and then fixed with 4% paraformaldehyde for 30 min at room temperature. Followed by washing with PBS three times and blocking with 1% BSA for 30 min at 37 °C, 1 × 1011 pfu representative clone of positive phage and IRP were added and incubated with the cells at 37 °C for 2 h. Cells were washed with PBS and incubated with anti-M13 antibody (Abcam, USA) at a dilution of 1:200 at 4 °C overnight. Subsequently, cells were washed with PBST and FITC Conjugated AffiniPure Goat Anti-rabbit IgG (working dilution of 1:100, Boster Biological Technology Co., Ltd., China) were added and incubated at 37 °C for 2 h. After washing three times with PBS, DAPI-Staining-solution (Boster Biological Technology Co., Ltd., China) was used to stain the nucleus. The cells were finally observed using an inverted microscope (SP8 STED 3X, Leica, Germany). IRP and PBS were used as control groups. The relative intensity of green fluorescence signal was calculated using Image J.
7
Stem Cell Adhesion on Titanium-based Surfaces
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As described in the previous step, BMSCs and VR-EPCs were seeded on the surfaces of UV-sterilized Ti, TNrs, and Fe3O4-TNrs, respectively, cells directly seeded in plates without sheets were set as control groups. Groups with the addition of SEMF were treated with 1 mT 15Hz SEMF for 1 h per day. The cells were fixed with 4% paraformaldehyde for 15 min and then washed three times with Immunol Staining Wash Buffer (Beyotime, Shanghai, China). Phalloidin (Sigma-Aldrich, USA) was diluted with Immunol Staining Primary Antibody Dilution Buffer (Beyotime, Shanghai, China) in a ratio of 1:100, and used for incubating the cells at room temperature for 60 min. DAPI staining solution (Boster, Wuhan, China) was then added and used for incubating the cells for 5 min. After washing three times, the cells were observed and photographed with a fluorescence microscope (EVOS FL Auto, Life Technologies, USA).
8
Magnetic Stimulation Enhances VR-EPC Adhesion
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VR-EPCs were seeded at a density of 1 × 104 cells/cm2 on the surfaces of Ti, TNrs, and Fe3O4-TNrs or into the plates wells without any sheets. VR-EPCs were cultured with M199 complete culture medium. The group with the addition of SEMF needed to be treated with 1 mT 15 Hz SEMF for 1 h per day. 3 days later, the cells were fixed with 4% paraformaldehyde for 15 min and then gently washed three times with Immunol Staining Wash Buffer (Beyotime, Shanghai, China). The cells were next treated with 1% BSA solution for 1 h, then incubated with NFATc1 primary antibody (ab25916, Abcam, MA, USA) diluted with Immunol Staining Primary Antibody Dilution Buffer (Beyotime, Shanghai, China) at 4 °C for 16 h. The cells were subsequently incubated at room temperature with Cy3 labeled secondary antibody (Boster, Wuhan, China) diluted with Immnol Fluorence Staining Secondary Antibody Dilution Buffer (Beyotime, Shanghai, China) for 1 h. After being gently washed, the cells were incubated with DAPI staining solution (Boster, Wuhan, China) for 5 min at room temperature. Then the cells could be observed and photographed under a fluorescence microscope.
9
Immunofluorescence Staining of Kidney, Ovary, and mTEC
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Immunofluorescence staining technique was performed as previously described.28 (link) Kidney sections, ovary sections and mTEC cells were stained with GLP-1R antibody (NBP1-97308, Novus Biologicals, USA) overnight at 4°C, followed by incubation with Alexa-488 goat anti-rabbit (#111-545-003, Jackson ImmunoResearch, USA). Then, the sections were counterstained with DAPI-staining-solution (Boster, USA), and the images were captured using inverted optical microscope (Nikon, Japan).
10
Histological Analysis of Mouse Heart Tissue
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Heart tissues of the above mice were fixed in 4% paraformaldehyde and embedded in paraffin. Cardiac tissue sections were stained with hematoxylin and eosin (H&E) to observe morphological changes and stained with Sirius red and Masson's trichrome to detect collagen deposition. Immunofluorescence staining of paraffin sections was carried out with 1:50 anti-HCN2 (Merck Millipore, Billerica, MA, USA) and further detected by fluorescently labeled secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA; Abcam, Cambridge, MA, USA), then paraffin sections were stained with DAPI-staining-solution (Boster, California, MA, USA).
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Images were acquired by fluorescence microscopy (ZEISS, Oberkochen, Germany) and analyzed by Image J. The representative images were repeated in at least three independent experiments.
This website utilizes technologies such as cookies to enable essential site functionality, as well as for analytics, personalization, and targeted advertising. To learn more, view the following link: Privacy Policy
Images were acquired by fluorescence microscopy (ZEISS, Oberkochen, Germany) and analyzed by Image J. The representative images were repeated in at least three independent experiments.
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