Fluoroshield with 4 6 diamidino 2 phenylindole

Manufactured by Merck Group

Fluoroshield with 4′,6-diamidino-2-phenylindole is a laboratory reagent used for staining and visualizing DNA in biological samples. It is a fluorescent dye that binds to the minor groove of double-stranded DNA, emitting a blue fluorescence when exposed to ultraviolet light.

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Lab products found in correlation

Tcs sp8 microscope, by Leica (1 mentions) Lsm 880 inverted confocal microscopy, by Zeiss (1 mentions) Bond max autostainer, by Leica (1 mentions) Anti ki67, by Abcam (1 mentions) Anti tff3, by Abcam (1 mentions)

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Fluoroshield (220 citations) Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI) (21 citations) Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (3 citations) Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI) histology mounting medium (2 citations)

3 protocols using fluoroshield with 4 6 diamidino 2 phenylindole

Fluorescent cGAMP Uptake Assay in Macrophages

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RAW264.7 and DC2.4 cells (5 × 10⁴) were cultured in the chambered coverslip with four wells. cGAMP^Fluo-loaded LNP was prepared by simply mixing of blank LNP and cGAMP^Fluo in water. The encapsulation rate of cGAMP^Fluo by 93-O17S-F was measured to be 85% by fluorescence spectrum. The free cGAMP^fluo or 93-O17S-F/cGAMP^Fluo was added to the medium at doses equivalent to cGAMP (200 ng/ml). After 4 hours of incubation, the medium was replaced and incubated by fresh medium containing LysoTracker Red DND-99 for another 1 hour. Then, the cells were washed by PBS and fixed in 4% paraformaldehyde solution for 10 min. Then, the slices were covered by Fluoroshield with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich). The images were captured using Leica TCS SP8 microscopes.

Chen J., Qiu M., Ye Z., Nyalile T., Li Y., Glass Z., Zhao X., Yang L., Chen J, & Xu Q. (2021). In situ cancer vaccination using lipidoid nanoparticles. Science Advances, 7(19), eabf1244.

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Quantifying Mitochondrial Morphology in HeLa Cells

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HeLa cells were cultured in eight-well chamber slides (Labtek, 155411) for 24 h prior to treatment. Cell membrane integrity was assessed with the amine-reactive dye LIVE/DEAD (L34958; Thermo Fisher Scientific) according to the manufacturer’s instructions. After fixation in 4% paraformaldehyde, cells were washed thrice in PBS and mounted with FluoroShield with 4,6-diamidino-2-phenylindole (Sigma-Aldrich, F6057). Images were captured with the Zeiss LSM 880 Inverted Confocal Microscopy using a 63× objective lens. Co-localization was assessed using Pearson’s correlation. Images were analyzed on ImageJ 1.53C NIH software for n ≥ 30 cells and presented as mean Rr ± standard deviation (SD). Mitochondrial area was measured using ImageJ Mito-Morphology Macro and normalized to total cellular area as described (Dagda et al., 2009 (link)). Mitochondrial network branching was quantified using Mitochondrial Network Analysis (MiNA) software as previously described (Valente et al., 2017 (link)).

Mohamud Y., Xue Y.C., Liu H., Ng C.S., Bahreyni A, & Luo H. (2021). Autophagy Receptor Protein Tax1-Binding Protein 1/TRAF6-Binding Protein Is a Cellular Substrate of Enteroviral Proteinase. Frontiers in Microbiology, 12, 647410.

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Immunofluorescence and Immunohistochemistry of Organoids

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Organoid cultures and tissues were fixed overnight in 10% formalin at room temperature. Paraffin-embedded organoids and tissues were serially sectioned into 10-μm slices. Paraffin sections were deparaffinized and rehydrated, followed by either staining for H&E or antigen retrieval in subboiling 10 mM sodium citrate buffer (pH 6.0) for 10 min. For IF, slides were permeabilized in 0.5% Triton X-100 in PBS and blocked in 1% goat serum in PBS for 30 min at room temperature. After blocking, slides were incubated with anti-Ki67 (1:200, Abcam) or anti-TFF3 (1:100, Abcam) overnight in a humidified chamber at 4°C. Sections were washed by PBS with 0.1% Tween 20 (PBST) (three times for 5 min each) and incubated with Alexa Fluor secondary antibodies (1:500) for 1 hour. After washing with PBST, slides were mounted with Fluoroshield with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich). IHC was performed using the automated BOND-MAX autostainer by Leica for the following antibodies: cytokeratin AE1/AE3 (1:200; Santa Cruz Biotechnology). Images were acquired with a Leica Inverted Confocal SP8 (Johns Hopkins Medicine Ross Imaging Center). Reagents used here are listed in table S2.

Williamson T., Gordon-Weeks P.R., Schachner M, & Taylor J. (1996). Microtubule reorganization is obligatory for growth cone turning. Proceedings of the National Academy of Sciences of the United States of America, 93(26), 15221-15226.

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