4 6 diamidino 2 phenylindole (dapi)

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DAPI (4',6-Diamidino-2-Phenylindole) is a fluorescent dye used in various laboratory applications. It binds strongly to adenine-thymine (A-T) rich regions in DNA, allowing for the visualization and detection of nucleic acids. DAPI is commonly used for nuclear staining and counterstaining in techniques such as fluorescence microscopy, flow cytometry, and DNA quantification.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Yol1 34, by Bio-Rad (1 mentions) Schneider s insect medium, by Thermo Fisher Scientific (1 mentions) Autoquant x, by Media Cybernetics (1 mentions) Concanavalin a coated, by Merck Group (1 mentions) Fetal bovine serum (fbs), by Developmental Studies Hybridoma Bank (1 mentions) Alexa fluor 568 and 647, by Thermo Fisher Scientific (1 mentions) Axio imager z1, by Zeiss (1 mentions) Orca r2, by Hamamatsu Photonics (1 mentions) Neurotrace fluorescent nissl stain, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions) Tcs sp8, by Leica (1 mentions) Prolong gold with dapi, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions) 24 well plate, by Thermo Fisher Scientific (1 mentions) Vectashield mounting medium, by Vector Laboratories (1 mentions)

4 protocols using 4 6 diamidino 2 phenylindole (dapi)

Imaging Cellular Structures in S2 Cells

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S2 cells transiently expressing CD8-GFP were grown on concanavalin A–coated (0.5 mg/ml; EMD Millipore) glass coverslips at 25°C in Schneider’s insect medium (Gibco) + 10% FBS (Invitrogen) and fixed with 4% paraformaldehyde for 10 min and subsequently extracted with 0.1% Triton X-100 for 10 min. After short washes in PBS and blocking with 10% FBS, cells were incubated with mouse anti-lamin Dm0 (clone ADL67, 1:50; Developmental Studies Hybridoma Bank) and rat anti–α-tubulin (YOL1/34, 1:100; Serotec), followed by short washes in PBS and incubation with Alexa Fluor 568 and 647 (1:1,000; Invitrogen). DNA was counterstained with DAPI (1 µg/ml; Sigma-Aldrich) before coverslips were mounted in 90% glycerol + 10% Tris, pH 8.5, + 0.5% N-propylgallate on glass slides. Images were acquired on an AxioImager Z1 (100×, Plan Apochromatic oil DIC objective lens, 1.4 NA; all from Carl Zeiss) equipped with a charge-coupled device (CCD) camera (ORCA-R2; Hamamatsu Photonics) using the Zen software (Carl Zeiss) and blind deconvolved using Autoquant X (Media Cybernetics). Images were processed (contrast adjustments) in Adobe Photoshop CS4 (Adobe) and represent either maximum intensity projections of deconvolved z stacks (step size: 0.22 µm; DAPI; α-tubulin) or a single slice (CD8-GFP; lamin Dm0).

Schweizer N., Pawar N., Weiss M, & Maiato H. (2015). An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region. The Journal of Cell Biology, 210(5), 695-704.

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Measuring FeSP Neuron Sizes in Brains

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The cell body and nuclear sizes of FeSP neurons were measured as described previously^{8 (link)}. In brief, whole female brains were fixed in 4% PFA, embedded in 5% agarose supplemented with 20% sucrose, and cut into 20 µm frozen sections in the coronal plane. Sections were incubated with a rabbit anti-Npb polyclonal antibody (RRID: AB_2810229), which has been shown to recognize Npba with high specificity^{13 (link)}, and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (RRID: AB_2534114; Thermo Fisher Scientific). Neuronal cell bodies and nuclei were stained with NeuroTrace Fluorescent Nissl Stain (NeuroTrace 530/615; Thermo Fisher Scientific) and DAPI (Roche Diagnostics), respectively. Fluorescent images were acquired with a confocal laser-scanning microscope (TCS SP8; Leica Microsystems), using excitation and emission wavelengths of 405 nm and 410–480 nm for DAPI, 488 nm and 495–545 nm for Alexa Fluor 488, and 552 nm and 620–700 nm for NeuroTrace 530/615.
The acquired images of the cell bodies and nuclei of Npb-immunoreactive neurons were converted to black and white binary images by thresholding using Adobe Photoshop (ver. 22; Adobe, San Jose, CA). The total areas of each cell body and nucleus were calculated by using ImageJ (https://imagej.nih.gov/ij/) and compared between genotypes.

Fleming T., Kikuchi Y., Nakajo M., Tachizawa M., Inazumi T., Tsuchiya S., Sugimoto Y., Saito D., Suyama M., Ohkawa Y., Baba T., Morohashi K.I, & Okubo K. (2022). Prostaglandin E2 receptor Ptger4b regulates female-specific peptidergic neurons and female sexual receptivity in medaka. Communications Biology, 5, 1215.

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Visualizing Muscimol Spread in Rat Brains

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After behavioral testing, rats were given an infusion (0.5 μl volume) of fluorophore-conjugated BODIPY TMR-X muscimol (Life Technologies, Carlsbad, CA) to visualize the spread of muscimol (Allen et al. 2008 (link)). The fluorescent muscimol was diluted to a concentration of 0.25 μg/μl in solution with equal parts PBS and vehicle DMSO. Twenty minutes after infusion of the fluorescent muscimol, rats were transcardially perfused with 0.9% saline followed by 10% buffered formalin. Brains were removed and allowed to sit in 10% buffered formalin for two days, and were then transferred to a 30% sucrose solution (30 mg sucrose/100 ml PBS). After sinking, the brains were sectioned (40 μm) with a cryostat and mounted on slides. Half the slides were stained with cresyl violet (0.5%) and photographed using a camera mounted microscope. The other half of the slides were stained with ProLong Gold with DAPI (Life Technologies, Carlsbad, CA), and visualized with a confocal microscope. Cannula placement and spread of the fluorescent muscimol was characterized by placing digital plates from the Paxinos and Watson (2005) rat brain atlas over pictures of the cresyl-stained and DAPI-stained brain slices using Adobe Illustrator.

Layfield D.M., Patel M., Hallock H, & Griffin A. (2015). Inactivation of the Nucleus Reuniens/Rhomboid Causes a Delay-dependent Impairment of Spatial Working Memory. Neurobiology of learning and memory, 125, 163-167.

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Immunofluorescence Staining of Cadherin and Selectin

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Cells (4×10⁴) were seeded on 24 well plates (Nunc) with coverslips on the bottom of each well and cultured for 24 h, until nearly confluent monolayers. Then cells were washed with PBS, fixed in ice-methanol for 20 min, and blocked with PBS-10% BSA for 30 min. For E-cadherin staining, cells were incubated with mAb against E-cadherin (clone 36, BD Biosciences; 1/200) and with secondary antibody Alexa Fluor 488 goat anti-mouse IgG (Invitrogen Life Technologies, 1/500). For double labeling, cells were incubated with mAb against SLe^x (clone KM93, Calbiochem; 1/60) and secondary antibody Alexa Fluor 594 goat anti-mouse IgM (Texas Red-conjugated; Invitrogen Life Technologies; 1/500). Finally, cells were washed, stained with DAPI (Sigma) and mounted with Vectashield mounting medium (Vector Laboratories). Antibodies were diluted in PBS-5% BSA, and incubated in a dark and humid chamber at room temperature. Separate images for E-cadherin, SLe^x and DAPI were captured digitally at 40× or 63× magnification. The green (for FITC), red (for Texas Red) and blue (for DAPI) components were merged and combined images were imported into Adobe Photoshop.

Bassagañas S., Carvalho S., Dias A.M., Pérez-Garay M., Ortiz M.R., Figueras J., Reis C.A., Pinho S.S, & Peracaula R. (2014). Pancreatic Cancer Cell Glycosylation Regulates Cell Adhesion and Invasion through the Modulation of α2β1 Integrin and E-Cadherin Function. PLoS ONE, 9(5), e98595.

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