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Dapi staining

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DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that binds to the minor groove of DNA. It is commonly used to stain and visualize cell nuclei in various applications such as fluorescence microscopy and flow cytometry.

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Lab products found in correlation

Trypsin inhibitor, by Thermo Fisher Scientific (2 mentions) Tryspin, by Thermo Fisher Scientific (2 mentions) Ab87625, by Abcam (2 mentions) Laminin, by Merck Group (2 mentions) Glutamine, by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions) Fluorescence microscope, by Leica (2 mentions) Arsenite, by Merck Group (2 mentions) Goat serum, by Vector Laboratories (2 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions) Nf200 antibody, by Merck Group (2 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Triton x 100, by Merck Group (2 mentions) Phosphate buffered saline (pbs), by Merck Group (2 mentions) Fugene 6, by Promega (2 mentions) Emccd camera, by Cytiva (1 mentions)

Close spelling variants of this product

4′,6-diamidino-2-phenylindole (DAPI) staining (4 citations) Vectashield mounting medium containing DAPI for nuclear staining (4 citations) Vectashield antifade mounting medium with DAPI for nuclear staining (3 citations) 4′,6-diamidino-2phenylindole (DAPI) staining (H-1800 (2 citations) DAPI-staining Mounting Medium (2 citations) DAPI staining mount (2 citations)

15 protocols using dapi staining

1

Immunofluorescence Analysis of Nop1 Localization

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Immunofluorescence analysis was performed as described previously (Wang et al., 2016). Briefly, 2 OD cells were fixed by addition of formaldehyde to a final concentration of 3.7% (v/v). Cells were pelleted, resuspended in 500 l of 3.7% formaldehyde in 0.1M potassium phosphate (pH 6.4) and fixed for 15 minutes at room temperature. Cells were washed 3× in in 0.1M potassium phosphate (pH 6.4) and once in sorbitol-citrate (128mM KH2P04, 36mM citrate, 1.2M sorbitol), followed by spheroplasting with zymolyase and glusulase in sorbitol-citrate. Spheroplasts were fixed on polylysine-coated slides, incubated with primary antibody overnight at 4°C, and secondary antibody for 4 hours at room temperature. Antibodies were diluted in PBS/BSA (50mM potassium phosphate, pH 7.4, 150mM NaCl, 1% bovine serum albumin, 0.1% sodium azide). Nop1 was detected using a mouse monoclonal Nop1 antibody (EnCor Biotechnology) at a 1:500 dilution and an anti-mouse fluorescein (FITC) antibody (Jackson ImmunoResearch) at 1:200. Nuclear DNA was detected using DAPI staining (Vectashield). Images were captured as z-stacks using a Delta Vision Elite System with EMCCD camera (Applied Precision, Issaquah, WA). Stacks were deconvolved, and compressed into a 2D image using SoftWoRX2.50 software (Applied Precision).
Mansisidor A., Molinar T J.r., Srivastava P., Dartis D.D., Delgado A.P., Blitzblau H.G., Klein H, & Hochwagen A. (2018). Genomic copy-number loss is rescued by self-limiting production of DNA circles. Molecular cell, 72(3), 583-593.e4.
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2

Senescence Evaluation by Cryosectioning

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After being fixed with formalin, samples were dehydrated with gradient sucrose solution and then frozen in Cryo-Gel (LeicaBiosystems, Richmond, IL, USA). Cryosectioning was accomplished through the use of Leica® CM1850 Cryostat (Mercedes Scientific, Lakewood Ranch, FL, USA) were frozen and subjected to cryosection. Cell senescence was assessed by β-galactosidase staining (SA-β-gal staining) using senescence β-Galactosidase Staining Kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s instructions. Additionally, DAPI staining (Vector Laboratories, Burlingame, CA, USA) was used to locate cell nuclei.
Wang N., He Y., Liu S., Makarcyzk M.J., Lei G., Chang A., Alexander P.G., Hao T., Padget A.M., de Pedro N., Menelaos T, & Lin H. (2021). Engineering Osteoarthritic Cartilage Model through Differentiating Senescent Human Mesenchymal Stem Cells for Testing Disease-Modifying Drugs. Science China. Life sciences, 65(2), 309-327.
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3

Neuroblastoma Cell Lines Cultivation

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Human MYCN-amplified NB cell lines SK-N-BE(2) (CRL-2271), IMR-32 (CCL-127) and CHP-212 (CRL-2273), and human MYCN-non-amplified NB cell lines SK-N-AS (CRL-2137) and SK-N-SH (HTB-11) were obtained from the American Type Culture Collection (ATCC, Virginia, United States) [44 (link), 45 ]. SK-N-BE(2) and CHP-212 cells were grown in a mixture (1:1) of Eagle’s Minimum Essential Medium (Sigma Aldrich by Merck, Darmstadt, Germany) and Ham´s F-12 Nutrient Mix (Gibco, California, United States). SK-N-AS cells were grown in Dulbecco’s Modified Eagle’s Medium (Gibco, California, United States) and 0.1 mM of Non-Essential Amino Acids (Gibco, California, United States). IMR-32 and SK-N-SH cells were grown in Eagle’s Minimum Essential Medium. 10% fetal bovine serum (FBS) (Gibco, California, United States) was added and the cell cultures were maintained at 37° C in a humidified atmosphere of 5% CO2. Cell lines were tested for Mycoplasma every three months by DAPI staining (Vector, California, United States). Cells passages lower than 20 were used in the described experiments.
Cuello H.A., Segatori V.I., Albertó M., Gulino C.A., Aschero R., Camarero S., Mutti L.G., Madauss K., Alonso D.F., Lubieniecki F, & Gabri M.R. (2018). Aberrant O-glycosylation modulates aggressiveness in neuroblastoma. Oncotarget, 9(75), 34176-34188.
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4

Granule Cell Migration Assay

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Cerebella from P7 pups were dissected in cold HBSS (Gibco), the meninges removed and were incubated in tryspin (Gibco) for 15 min at 37 °C. The reaction was then stopped by adding trypsin inhibitor (Life Technologies) and incubating at room temperature for 5 min. Cerebella were triturated and purified on an Optiprep gradient [93 (link)]. Cells were plated in DMEM (Gibco) supplemented with 0.5-mM glutamine (Gibco). Cell migration was assessed using an 8.0-μm pore polycarbonate membrane Transwell inserts (Corning Life Sciences, Acton, MA). The lower surface of the insert was pre-coated with laminin (Sigma). Granule cell suspensions (20 × 104 cells) were added to the upper compartment, and 600-μl culture medium was added to the lower compartment with recombinant Gpi1 (Abcam ab87625) or vehicle (water). After a 24-h incubation at 37 °C, the membranes were fixed with 4% paraformaldehyde for 10 min at room temperature and washed with PBS. Cells on the upper surface of the membrane were swiped with cotton swabs and cells that invaded through the membrane to the under membrane were visualised by DAPI staining (VectorLab) and counted using a fluorescence microscope (Leica).
Finelli M.J., Paramo T., Pires E., Ryan B.J., Wade-Martins R., Biggin P.C., McCullagh J, & Oliver P.L. (2018). Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase. Molecular Neurobiology, 56(3), 1558-1577.
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5

Immunohistochemical Analysis of Brain Sections

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Mice were anesthetized (ketamine/xylazine, 150 mg/kg, 12 mg/kg body weight) and perfused transcardially with 4 % paraformaldehyde. Brains were then cyroprotected in a 30 % sucrose solution overnight. 30 μm coronal sections were cut with a microtome (Microm HM 430, Thermo Scientific) and blocked in a solution consisting 4 % normal goat serum, 1% BSA and 0.3 % Tritonx100 in 1× PBS for 1 hour at room temperature. They were soon after incubated overnight at 4 °C with primary antibodies (1:1000 chicken anti-GFP, Aves Labs; 1:1000 mouse anti-GAD67, Millipore) diluted in a blocking solution. After washing, sections were then incubated in Alexa Fluor–conjugated secondary antibodies (1:1000 anti-chicken 488; 1:1000 anti-mouse 594, Invitrogen) for 2 hours at room temperature. Slices were then mounted with a mounting medium for DAPI staining (Vector Laboratories) and imaged using a fluorescence microscope (ApoTome.2, Zeiss, Figure S2A and B).
Makino H., Ren C., Liu H., Kim A.N., Kondapaneni N., Liu X., Kuzum D, & Komiyama T. (2017). Transformation of Cortex-wide Emergent Properties during Motor Learning. Neuron, 94(4), 880-890.e8.
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6

Apoptosis Evaluation in Colon Cancer Cell Lines

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HT29-s and HCT-15-s were treated with or without MLT and AGP for 48 h. Apoptotic features were evaluated using DAPI staining (Vector Laboratories, Burlingame, CA) as described earlier14 (link).
Sokolov D., Sharda N., Giri B., Hassan M.S., Singh D., Tarasiewicz A., Lohr C., von Holzen U., Kristian T., Waddell J., Reiter R.J., Ahmed H, & Banerjee A. (2022). Melatonin and Andrographolide synergize to inhibit the colospheroid phenotype by targeting Wnt/beta-catenin signaling. Journal of pineal research, 73(1), e12808.
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7

Immunofluorescence Staining Protocol

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IF was performed as previously described (Manku et al., 2016 (link)). Briefly, slides were first dewaxed and rehydrated using Citrisol and Trilogy solution (Cell Marque, Rocklin, CA). Following treatment with DAKO Target Antigen Retrieval Solution (Agilent Technologies Inc., Santa Clara, CA), the sections were incubated with PBS containing 10% BSA and 10% donkey serum for one hour to block non-specific protein interactions. The sections were then incubated with anti-Aquaporin 1 (AQP1) antibody (ab189291, Abcam, Cambridge, UK) or anti-POD1 (Tcf21) (bs-8688r, Bioss Antibodies, Woburn, MA) antibody diluted in PBS containing 10% BSA, 0.1% Triton-X 100, and donkey serum overnight at 4°C. Once the overnight incubation was complete, slides were incubated with a secondary goat anti-rabbit Alexa Flour 488 antibody (Invitrogen, Waltham, MA) diluted in PBS containing 1% BSA for one hour at room temperature. Nuclear staining was performed using DAPI staining (Vector Labs, Burlingame, CA) for 5 minutes. The slides were then mounted with one drop of room temperature Fluoromount-G (Electron Microscopy Sciences) and cover-slipped. Slides were imaged on the Cytation 5 Cell Imaging Multi-Mode Reader (Biotek, Winooksi, VT, USA).
Tran-Guzman A., Moradian R., Walker C., Cui H., Corpuz M., Gonzalez I., Nguyen C., Meza P., Wen X, & Culty M. (2022). Toxicity profiles and protective effects of antifreeze proteins from insect in mammalian models. Toxicology letters, 368, 9-23.
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8

Neurite growth and cell death analysis

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Neuronal Neuro2a (N2a) and HeLa cells were cultures in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with glutamax, 1% penicillin-streptomycin, and 10% fetal calf serum (all Gibco). Cells were co-transfected with Fugene 6 (Promega) for 48 h as per the manufacturer’s protocol. shRNA constructs were from Sigma (#11857 for Gpi1 and as previously described for Oxr1 [14 (link)]). For neurite growth assay, medium was replaced with serum-free medium when transfecting and incubated for 48 h. For cell death assay, cells were treated with 250-μM arsenite (Sigma) for 4 h. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS (Sigma) twice, and blocked with blocking buffer (5% goat serum (VectorLab), 0.5% Triton X-100 (Sigma)) for 1 h at room temperature. Neurites and pyknotic nuclei were visualised with NF200 antibody (Sigma N4142) and DAPI staining (Vectorlabs), respectively.
Finelli M.J., Paramo T., Pires E., Ryan B.J., Wade-Martins R., Biggin P.C., McCullagh J, & Oliver P.L. (2018). Oxidation Resistance 1 Modulates Glycolytic Pathways in the Cerebellum via an Interaction with Glucose-6-Phosphate Isomerase. Molecular Neurobiology, 56(3), 1558-1577.
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9

Immunohistochemical Analysis of 3D Spheroid Cocultures

Cited 2 times
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The 3D spheroid cocultures were kept in 4% paraformaldehyde at 4 °C for 30 min. The specimens were then processed, embedded in paraffin and sectioned at 5 μm, essentially as we did before [60 (link), 61 (link)]. Heat-induced antigen retrieval was performed in citrate buffer using a pressure cooker. Sections were incubated with primary antibodies overnight at 4 °C. Primary antibodies used in this study were P2RY12 (BioLegend, CA) and Nestin (Rat401, DSHB) as we used in previous studies. Sections were then incubated with FITC- or TRICT-conjugated IgG (for 1 h at room temperature), followed by DAPI staining (Vector Laboratories). Images were acquired with Olympus BX41 microscope. Six random fields from three different tissues in each group were quantified using the ImageJ software.
Peng W.T., Banta L.M., Charles T.C, & Nester E.W. (2001). The chvH Locus of Agrobacterium Encodes a Homologue of an Elongation Factor Involved in Protein Synthesis. Journal of Bacteriology, 183(1), 36-45.
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10

HUVEC Cell Growth Monitoring

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Cell growth was monitored using either DAPI staining (Vector Labs) or the alamarBlue® (Invitrogen Co.) assay as described in the manufacturer’s instructions. For the 96-well plate growth assay, HUVEC were plated at a range of 1x103 to 5x103 cells/mL. After treatments, alamarBlue® reagent was added at 10% the total well volume. Fluorescence readings at 560 and 590 nm were taken at time intervals of 2, 4, 8, 12, 18, 24, and 48 hours post treatment.
Kunkler B., Salamango D., DeBruine Z.J., Ploch C., Dean S., Grossens D., Hledin M.P., Marquez G.A., Madden J., Schnell A., Short M, & Burnatowska-Hledin M.A. (2018). CUL5 is required for thalidomide-dependent inhibition of cellular proliferation. PLoS ONE, 13(5), e0196760.
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