Fluoroshield solution with dapi

Manufactured by Merck Group

Sourced in Macao

Fluoroshield solution with DAPI is a laboratory reagent used for staining and preserving biological samples. It contains the fluorescent dye DAPI (4',6-diamidino-2-phenylindole) which binds to DNA, allowing for the visualization of cell nuclei. The solution is designed to maintain the sample's structure and fluorescence over time.

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Lab products found in correlation

Erythrocyte lysis buffer, by Thermo Fisher Scientific (1 mentions) Apotome microscope, by Zeiss (1 mentions) Anti srbc antibody, by Merck Group (1 mentions) Pkh26 dye, by Merck Group (1 mentions) Goat serum, by Abcam (1 mentions) Texas red, by Thermo Fisher Scientific (1 mentions) Goat anti mouse alexa fluor 488, by Abcam (1 mentions) Mouse anti human cd31, by Abcam (1 mentions) Cybershot, by Sony (1 mentions) Dulbecco modified eagle medium (dmem), by LGC (1 mentions)

3 protocols using fluoroshield solution with dapi

Phagocytosis Assay for CD31 Knockdown PBM

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For phagocytosis assays in PBM after CD31 knockdown, fluoresceinated, antibody-coated sheep red blood cells (SRBCs) were used. Briefly, SRBCs (Colorado serum company, USA) were labeled with PKH26 dye (Sigma-Aldrich, MO) according to manufacturer protocol, then opsonized with anti-SRBC antibody (Sigma-Aldrich, MO). Non-opsonized, fluoresceinated SRBCs were used as controls.
CD31 knockdown or control PBM were incubated with 1 μl of pelleted SRBCs for 45 minutes at 37 ℃. SRBCs that were not ingested were removed through hypotonic lysis with erythrocyte lysis buffer (eBiosciences, ThermoFisher Scientific, IL) and washed with PBS. Samples were then fixed with 1% paraformaldehyde. The samples were blinded and then analyzed using bright field and fluorescence microscopy using an Olympus DP71. Results are shown as phagocytic index, defined as the total number of SRBCs phagocytized by 100 monocytes. Representative pictures were taken from PBM fixed after the phagocytic assay and mounted in Fluoroshield solution with DAPI (Sigma-Aldrich, MO). Figures shown were taken as light field using a Zeiss Apotome microscope.

Merchand-Reyes G., Robledo-Avila F.H., Buteyn N.J., Gautam S., Santhanam R., Fatehchand K., Mo X., Partida-Sanchez S., Butchar J.P, & Tridandapani S. (2019). CD31 acts as a checkpoint molecule and is modulated by FcγR-mediated signaling in monocytes. Journal of immunology (Baltimore, Md. : 1950), 203(12), 3216-3224.

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Monocyte Attachment and Localization Assay

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Freshly-isolated PBM were incubated with polymyxin B and the indicated inhibitors for 45 minutes prior to ΔIgG stimulation at a density of 4×10⁶ cells/mL. After 4 hours, 2×10⁵ were seeded on poly-L-lysine coated coverslips for 15 minutes at 37 °C to allow attachment. Monocytes were fixed with 4% paraformaldehyde overnight at 4 °C, then blocked with 10% goat serum (Abcam, MA) for 1 hour at room temperature under permeabilized (0.1% Triton X-100) or non-permeabilized conditions. Cells were stained with mouse anti-human CD31 (Abcam, MA) overnight and goat anti-mouse Alexa Fluor 488 (Abcam, MA) plus wheat germ agglutinin (WGA) labeled with Texas red (ThermoFisher Scientific, IL) for 1 hour. Finally, coverslips were mounted with Fluoroshield solution with DAPI (Sigma-Aldrich, MO). Images were acquired using a Zeiss LSM800 confocal microscope, and then analyzed using ImageJ (NIH, USA).

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Micronucleus Assay for Genotoxicity Evaluation

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In 24-well plates, FMM-1 were grown in DMEM (LGC Biotechnology) medium supplemented with 10% fetal bovine serum (SFB) (Invitrogen) where 2 x 10 4 cells / well were plated and incubated at 37 °C with 5% CO2 for 24 h. Then, the cells were exposed to A. satureioides extracts diluted in DMEM + 10% SFB and to ethyl methane sulfanate (EMS); 5 mM as a positive control group for 24 h.
Later, the cells were fixed with 4% formaldehyde. Then, 500 µL of PBS and 1 drop of Fluoroshield solution with DAPI (Sigma-Aldrich) were added to the wells, which were photographed with a digital camera (Sony F828 digital, CyberShot, 8.0 megapixels) coupled with an inverted light microscope. At least 10 photos per well were taken and the number of micronuclei was determined to be 2,000 cells / well. DNA structures contained in the cytoplasm separated from the main nucleus were identified as micronuclei, with an area less than 1/3 of the main nucleus area. Cells in mitosis and that exhibited nuclear fragmentation by apoptosis were not considered in the count (Oliveira et al., 2017; (link)Sousa et al., 2020) (link).

Ferreira E.C.C., Gonçalves T.M., Pereira T.C., Hasna A.A., Oliveira F.E.d., Jorjão A.L., Camargo S.E.A., Oliveira L.D.d., & Spalding M. (2021). The biocompatibility of Achyrocline satureioides plant extract over human gingival fibroblasts. Research Society and Development, 10(1), e37610111902-e37610111902.

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