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Dapi for nuclear counterstaining

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DAPI is a fluorescent dye used for nuclear counterstaining in various biological applications. It binds strongly to adenine-thymine (A-T) rich regions of DNA, emitting blue fluorescence when excited by ultraviolet (UV) or violet light. DAPI provides a simple and effective way to visualize and quantify cell nuclei in a sample.

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Lab products found in correlation

Bovine serum albumin (bsa), by Corning (2 mentions) Normal goat serum (ngs), by Jackson ImmunoResearch (2 mentions) Anti pan cytokeratin mouse monoclonal antibody, by BioLegend (2 mentions) Qdot 800, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Alexa fluor 647, by Thermo Fisher Scientific (2 mentions) Rabbit anti c peptide, by Cell Signaling Technology (1 mentions) Tcs sp8, by Leica (1 mentions) Anti insulin, by Cell Signaling Technology (1 mentions) Anti foxo1, by Cell Signaling Technology (1 mentions)

4 protocols using dapi for nuclear counterstaining

1

Multicolor Immunostaining of CTC Samples

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Enriched CTC cell preparations were plated on coverslips and fixed with pre-warmed (37 °C) 2% formaldehyde (Sigma-Aldrich, St. Louis, MO) in 1× PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2) for 15 min and blocked overnight in 10% Normal Goat Serum (Jackson ImmunoResearch, West Grove, PA) plus 6% Bovine Serum Albumin (BSA) in PBS (Corning, Tewksbury, MA).
Cells were subsequently immunostained for: (1) the leukocyte marker CD45 using an anti-CD45 antibody directly conjugated with QDot-800 (Invitrogen, Waltham, MA Catalog # Q10156); (2) the epithelial marker cytokeratin, using an anti-pan-cytokeratin mouse monoclonal antibody recognizing human cytokeratins 4, 5, 6, 8, 10, 13, and 18 (BioLegend, San Diego, CA catalog # 628602) conjugated to CF594 using Biotium Mix-n- Stain antibody labeling kit (catalog # 92236); (3) tubulin (Novus rat anti-tyrosinated tubulin, Catalog # NB600-506), followed by goat anti-rat IgG secondary antibody, AlexaFluor 647 (Thermo Fisher Scientific, catalog # A21247); (4) androgen receptor AR [Rabbit anti-AR (N-terminal) catalog # ab3510], and goat anti-rabbit IgG secondary antibody, AlexaFluor 488 (Catalog # A11034); and (5) DAPI for nuclear counterstaining (Invitrogen). Coverslips were mounted on a glass slide by using Mowiol mounting media (Electronic Microscopy Sciences, Hatfield, PA).
Mukhtar E., Worroll D., Galletti G., Schuster S., Piha-Paul S.A, & Giannakakou P. (2020). Quantitative analysis of taxane drug target engagement of microtubules in circulating tumor cells from metastatic castration resistant prostate cancer patients treated with CRXL301, a nanoparticle of docetaxel. Cancer Drug Resistance, 3(3), 636-646.
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2

Multicolor Immunostaining of CTC Samples

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Enriched CTC cell preparations were plated on coverslips and fixed with pre-warmed (37 °C) 2% formaldehyde (Sigma-Aldrich, St. Louis, MO) in 1× PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2) for 15 min and blocked overnight in 10% Normal Goat Serum (Jackson ImmunoResearch, West Grove, PA) plus 6% Bovine Serum Albumin (BSA) in PBS (Corning, Tewksbury, MA).
Cells were subsequently immunostained for: (1) the leukocyte marker CD45 using an anti-CD45 antibody directly conjugated with QDot-800 (Invitrogen, Waltham, MA Catalog # Q10156); (2) the epithelial marker cytokeratin, using an anti-pan-cytokeratin mouse monoclonal antibody recognizing human cytokeratins 4, 5, 6, 8, 10, 13, and 18 (BioLegend, San Diego, CA catalog # 628602) conjugated to CF594 using Biotium Mix-n- Stain antibody labeling kit (catalog # 92236); (3) tubulin (Novus rat anti-tyrosinated tubulin, Catalog # NB600-506), followed by goat anti-rat IgG secondary antibody, AlexaFluor 647 (Thermo Fisher Scientific, catalog # A21247); (4) androgen receptor AR [Rabbit anti-AR (N-terminal) catalog # ab3510], and goat anti-rabbit IgG secondary antibody, AlexaFluor 488 (Catalog # A11034); and (5) DAPI for nuclear counterstaining (Invitrogen). Coverslips were mounted on a glass slide by using Mowiol mounting media (Electronic Microscopy Sciences, Hatfield, PA).
Mukhtar E., Worroll D., Galletti G., Schuster S., Piha-Paul S.A, & Giannakakou P. (2020). Quantitative analysis of taxane drug target engagement of microtubules in circulating tumor cells from metastatic castration resistant prostate cancer patients treated with CRXL301, a nanoparticle of docetaxel. Cancer Drug Resistance, 3(3), 636-646.
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3

Immunostaining of Eye Sections

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For immunostaining, eye sections were equilibrated to room temperature, washed, blocked, and incubated with the primary antibodies, goat anti m-leptin (R&D Systems, Minneapolis, MN, USA) and rabbit anti-C-peptide (Cell Signaling, Danvers, MA, USA) in the presence of 0.1% Triton X-100 and 10% serum. After washing, secondary antibodies, anti-goat Alexa633, and anti-rabbit Alexa594, respectively (Thermo Fisher Scientific, Waltham, MA, USA), were applied, and mounting with medium containing DAPI for nuclear counterstaining (Thermo Fisher Scientific, Waltham, MA, USA) was performed after repeated washing. Imaging was performed using a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany) with the following settings: DAPI excitation 405 nm, detection 450–470 nm; ZsGreen excitation 488 nm, detection 500–525 nm; mCherry excitation 548 nm, detection 560–580 nm; Alexa 594 (C-peptide staining) excitation 594 nm, detection 600–620 nm and Alexa 633 (leptin staining) excitation 633 nm, detection 640–680 nm. To avoid spectral overlap imaging was performed using in-between-frames sequential imaging.
Leibiger B., Moede T., Valladolid-Acebes I., Paschen M., Visa M., Leibiger I.B, & Berggren P.O. (2021). Ectopic Leptin Production by Intraocular Pancreatic Islet Organoids Ameliorates the Metabolic Phenotype of ob/ob Mice. Metabolites, 11(6), 387.
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4

Immunostaining of Pancreatic Tissue

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For immunostaining, pancreas tissue sections were equilibrated to room temperature, washed, blocked, and then, incubated with the primary antibodies (anti-FoxO1 and anti-insulin; Cell Signaling Technology, Danvers, MA, USA) in the presence of 0.1% Triton X-100 and 10% serum. After washing, secondary antibodies were applied, and mounting with medium containing DAPI for nuclear counterstaining (Thermo Fisher Scientific) was performed after repeated washing.
Paschen M., Moede T., Valladolid-Acebes I., Leibiger B., Moruzzi N., Jacob S., García-Prieto C.F., Brismar K., Leibiger I.B, & Berggren P.O. (2018). Diet-induced β-cell insulin resistance results in reversible loss of functional β-cell mass. The FASEB Journal, 33(1), 204-218.
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