4 6 diamidino 2 phenylindole (dapi)

Manufactured by Leica Microsystems

Sourced in Germany, United States

DAPI is a fluorescent stain used to label DNA in biological samples. It binds to the minor groove of double-stranded DNA, emitting blue fluorescence when excited by ultraviolet light. DAPI is commonly used in microscopy techniques to visualize cell nuclei and chromosomes.

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22 protocols using 4 6 diamidino 2 phenylindole (dapi)

Multimodal Imaging of Tissue Sections

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Tissue sections stained by IHC were scanned by an Aperio CT scanner (Aperio Technologies) with a ×20 objective. Digital images were saved on the eSlide Manager database (version 12.3.2.5030). Images were manually inspected, analyzed, and annotated using the ImageScope software (Aperio Technologies).
Fluorescent images were taken with a Leica DM5500B microscope (Leica Microsystems) using a ×20 objective. We used the following fluorescent filters from Leica Microsystems: 4,6-diamidino-2-phenylindole for blue fluorescence (excitation 360 nm, excitation 470 nm), L5 for green fluorescence (excitation 480 nm, excitation 527 nm), and TXR for red fluorescence (excitation 560 nm, excitation 630 nm). Image acquisition was performed using the Leica Application Suite X (LAS X).

Romero-Moreno R., Curtis K.J., Coughlin T.R., Miranda-Vergara M.C., Dutta S., Natarajan A., Facchine B.A., Jackson K.M., Nystrom L., Li J., Kaliney W., Niebur G.L, & Littlepage L.E. (2019). The CXCL5/CXCR2 axis is sufficient to promote breast cancer colonization during bone metastasis. Nature Communications, 10, 4404.

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Immunofluorescence Imaging of Treated Cells

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BC cells cultured on circular glass discs in the wells of 24-well plates were exposed to 20 μM of BA for 24 h and/or 15 ng/ml of TNF-α for 4 h. Then, the cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 1% Triton X-100 for 15 min, washed three times with ice-cold PBS, and blocked with 5% bovine serum albumin for 1 h. Following overnight incubation with primary antibodies at 4°C, the cells were washed with ice-cold PBS and treated with fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G secondary antibody (dilution, 1:500; Beyotime Institute of Biotechnology) for 1 h at room temperature. Finally, the cells were stained with 4′,6-diamidino-2-phenylindole (dilution, 1:10,000) for 10 min and photographed with the use of a confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany).

Zeng A., Liang X., Zhu S., Liu C., Luo X., Zhang Q, & Song L. (2020). Baicalin, a Potent Inhibitor of NF-κB Signaling Pathway, Enhances Chemosensitivity of Breast Cancer Cells to Docetaxel and Inhibits Tumor Growth and Metastasis Both In Vitro and In Vivo. Frontiers in Pharmacology, 11, 879.

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Chromosome Spread Analysis by Fluorescence Microscopy

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Slides with chromosome spreads were analysed using a fluorescence microscope, Leica DM 4000, equipped with a monochrome digital camera DFC350 FX and filter cubes corresponding to fluorochromes Alexa488, Cy3, and DAPI (Leica-Microsystems). The LAS X core computer program was used to obtain and process colour images.
Analysis of the 3D morphology of the gonads and whole-mount FISH was performed by laser scanning confocal microscopy (using a Leica TCS SP5 based on the inverted microscope Leica DMI 6000 CS, Leica-Microsystems). Nuclei were scanned in XYZ planes using lens HC PL APO 40×. Images were obtained by the LAS AF program (Leica-Microsystems, Germany).

Dedukh D., Litvinchuk J., Svinin A., Litvinchuk S., Rosanov J, & Krasikova A. (2019). Variation in hybridogenetic hybrid emergence between populations of water frogs from the Pelophylax esculentus complex. PLoS ONE, 14(11), e0224759.

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Uptake Kinetics of Labeled Liposomes

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Cells were plated 24–48 h before each experiment, on sterile 22 mm coverslips inside 6-well plates at a density of 5 × 10⁴/mL. Following attachment, the cells were exposed to DiI-labelled liposomes (L1, L2, and L3) for various time intervals at 37 °C in the incubator. The employed liposomal dilution had an arsenic concentration of 18 µmol/L. After the intended time period, the spent media were removed, and cells were washed three times with PBS and fixed with PBS-buffered 4% paraformaldehyde at 25 °C for 8 min. The coverslips were again washed with PBS and mounted on a slide using DAPI containing anti-fade ProLong Gold reagent. The fluorescence emitted from each slide was observed via a fluorescent confocal microscope at 570 nm and 460 nm for DiI and DAPI, respectively (Leica Microsystems, Wetzlar, Germany).

Akhtar A., Ghali L., Wang S.X., Bell C., Li D, & Wen X. (2019). Optimisation of Folate-Mediated Liposomal Encapsulated Arsenic Trioxide for Treating HPV-Positive Cervical Cancer Cells In Vitro. International Journal of Molecular Sciences, 20(9), 2156.

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Immunostaining Protocol for PKCδ and Cdc25B in Embryos

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Immunostaining was performed as previously described (28 (link)). PKCδ was detected using anti-PKCδ (1:200; cat no. 610398; BD Biosciences) at 4°C overnight. Subsequently, the enbyors were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody (1:100; cat. no. AP130F; Chemicon International, Inc., Temecula, CA, USA) at room temperature for 2 h. Chromosomes were labeled with 10 g/ml DAPI (cat. no. P36931; Invitrogen; Thermo Fisher Scientific, Inc.). For PKCδ and Cdc25B double staining, PKCδ was detected using anti-PKCδ and FITC-conjugated goat anti-mouse IgG secondary antibody. Following PKCδ immunostaining, embryos were washed three times with PBS and Cdc25B was detected using anti-Cdc25B (1:200; cat. no. sc-326; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h, followed by tetrarhodamine isothiocyanate-conjugated rabbit anti-goat IgG secondary antibody (1:200; cat. no. ab7087; Abcam,) at room temperature for 2 h. Following secondary incubation, the embryos were stained with DAPI (10 g/ml) for 10 min at room temperature and observed under a confocal laser-scanning microscope (magnification, ×400; Leica Microsystems GmbH, Wetzlar, Germany).

Liu Y., Deng X., Wu D., Jin M, & Yu B. (2019). PKCδ promotes fertilization of mouse embryos in early development via the Cdc25B signaling pathway. Experimental and Therapeutic Medicine, 18(5), 3281-3290.

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Spindle Staining in Oocytes

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To stain the spindles, oocytes were fixed in 4% (w/v) paraformaldehyde in DPBS (pH 7.4) for 30 min, and permeabilized in 0.5% Triton X-100 for 5 min. Then, oocytes were incubated with 2 μg/ml anti-α-Tubulin (Millipore, Billerica, MA) for 1 h, washed three times for 5 min in DPBS and stained with 4’,6-diamidino-2-phenylindole (DAPI, Life Technologies, ThermoFisher Scientific, Waltham, MA) prior to being examined using Leica SP5 spectral scanning confocal microscope (Leica Microsystem, Buffalo Grove, IL) using excitation at 488 nm and emission at 530 nm (α-Tubulin), and excitation at 350 nm and emission at 470 nm (DAPI).

Zhang M., Bener M.B., Jiang Z., Wang T., Esencan E., Scott R I.I.I., Horvath T, & Seli E. (2019). Mitofusin 2 plays a role in oocyte and follicle development, and is required to maintain ovarian follicular reserve during reproductive aging. Aging (Albany NY), 11(12), 3919-3938.

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Apoptosis Detection via TUNEL Assay

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Apoptosis was detected using the TUNEL assay, according to the guidelines of the manufacturer (CF^TM488A TUNEL Assay Apoptosis Detection Kit, Biotium, Hayward, CA, USA). The cells were grown on glass coverslips and then treated with derivative 2. After paraformaldehyde fixation, the TUNEL reaction mixture containing the terminal deoxynucleotidyl transferase (TdT) enzyme was added, as reported by Iacopetta et al. [62 (link),63 (link)]. Samples were washed, incubated with DAPI (Sigma; 0.2 µg/mL) for nuclei counterstaining, and then observed and imaged under a fluorescence microscope (Leica DM6000, Leica Microsystems GmbH, Wetzlar, Germany; magnification ×20, λ_ex/em maxima of 490/515 nm for CF^TM488A or 350/460 nm for DAPI). Images are representative of three independent experiments.

Chimento A., Santarsiero A., Iacopetta D., Ceramella J., De Luca A., Infantino V., Parisi O.I., Avena P., Bonomo M.G., Saturnino C., Sinicropi M.S, & Pezzi V. (2021). A Phenylacetamide Resveratrol Derivative Exerts Inhibitory Effects on Breast Cancer Cell Growth. International Journal of Molecular Sciences, 22(10), 5255.

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Immunolocalization of Epac and Rap1 in Mouse Oocytes

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Mouse oocytes were fixed with 2% formaldehyde for 15 min at 37°C and permeabilized with 0.02% Triton X-100 for 1 h at 37°C. To determine the expression of Epac and Rap1, the fixed oocytes were incubated with goat anti-Epac1 (sc-8879; Santa Cruz Biotechnology, Inc.) polyclonal antibodies and rabbit anti-Rap1 (sc-65; Santa Cruz Biotechnology, Inc.) polyclonal antibodies for 1 h at 37°C. After being washed with PBS, oocytes were transferred to a solution with secondary antibodies: Cy3-conjugated anti-goat IgG (1:500; cat. no. 705–165-003; Jackson ImmunoResearch Laboratoris, Inc., West Grove, PA, USA) and Cy3-conjugated anti-rabbit IgG (1:500; cat. no. 111-165-003; Jackson ImmunoResearch Laboratoris, Inc.) for 1 h at 37°C. All of the secondary antibodies were purchased from Jackson Laboratories (Bar Harbor, ME, USA). DAPI (0.2 µg/ml, DA0001, Sigma-Aldrich) was used to stain the nuclei for 5 min at RT. Slides were examined using a laser-scanning confocal microscope with a 63 × oil immersion objective lens and a Leica SP2 krypton-argon ion laser (Leica Microsystems, Wetzlar, Germany) for the simultaneous excitation of fluorescence for proteins and DAPI for DNA.

Wang J.C., Geng Y., Han Y., Luo H.N, & Zhang Y.S. (2018). Dynamic expression of Epac and Rap1 in mouse oocytes and preimplantation embryos. Experimental and Therapeutic Medicine, 16(2), 523-528.

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Cellular Uptake and DOXO Release

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Particle uptake and DOXO release inside cells
were also followed by fluorescence microscopy. HeLa cells were seeded
on poly-l-lysine coated glass coverslips (12 × 12 mm², Sigma-Aldrich) placed inside 6-well plates (2 × 10⁵ cells per well) with 2 mL of DMEM and grown for 24 h at standard
culture conditions. Then, PSS/PLL/DOXO/HA-coated and (PSS/PLL/DOXO)₂/HA-coated GNRs (2.5 × 10¹⁰ NP/mL, 250 μL)
were added to cells. After 6 h of incubation, the cells were washed
three times with PBS and fresh medium was added. Next, some cells
were irradiated with a CW 808 nm fiber-coupled diode laser source
(50 W, Oclaro Inc., San Jose, CA) for 5 min at 0.5 W/cm². After the desired incubation time (4, 6, 8, 12, and 24 h), the
cells were washed three times with PBS, fixed with 4% (w/v) paraformaldehyde
for 10 min, washed again with PBS, treated with Triton X-100 for 10
min, and, finally, washed again with PBS. Then, the coverslips were
mounted on glass slides, stained with DAPI (Invitrogen, USA) and cured
for 24 h at −20 °C. The samples were visualized at 63×
using a wide field fluorescence inverted microscope (Leica DMI6000B,
Leica Microsystems, Germany) using the blue channel for DAPI (λ_ex = 350 nm), the red channel for DOXO (λ_ex = 520 nm), and transmitted light in differential interference contrast
mode.

Villar-Alvarez E., Cambón A., Pardo A., Mosquera V.X., Bouzas-Mosquera A., Topete A., Barbosa S., Taboada P, & Mosquera V. (2018). Gold Nanorod-Based Nanohybrids for Combinatorial Therapeutics. ACS Omega, 3(10), 12633-12647.

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DAPI Staining of hDMSCs on GelMA-KerMA

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To evaluate the attachment of hDMSCs on GelMA-KerMA patches, DAPI staining was undertaken on day 7 of culture. First, the growth medium was discarded, and patches were rinsed three times with 100 μL of pre-warmed PBS. After that, the cells were fixed with 4% formaldehyde (Sigma–Aldrich, Darmstadt, Germany) at room temperature (RT) for 30 min, rinsed with PBS, and permeabilized in 0.1% Triton X-100 (Merck KGaA, Darmstadt, Germany) at RT for 10 min. Next, the samples were incubated in 1 μg/mL DAPI (Invitrogen, Waltham, MA, USA) for 20 min at RT to stain the nuclei of the cells. Finally, the DAPI solution was removed, and patches were observed under an inverted fluorescence microscope (Leica Microsystems Inc., Deerfield, IL, USA).

Bedir T., Baykara D., Yildirim R., Calikoglu Koyuncu A.C., Sahin A., Kaya E., Tinaz G.B., Insel M.A., Topuzogulları M., Gunduz O., Ustundag C.B, & Narayan R. (2024). Three-Dimensional-Printed GelMA-KerMA Composite Patches as an Innovative Platform for Potential Tissue Engineering of Tympanic Membrane Perforations. Nanomaterials, 14(7), 563.

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