Vectashield antifade with dapi

Manufactured by Vector Laboratories

Sourced in United States

VECTASHIELD Antifade with DAPI is a mounting medium designed to reduce photobleaching and enhance fluorescence signals. It contains the fluorescent dye DAPI, which binds to DNA and emits blue fluorescence.

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Lab products found in correlation

Cytospin 4, by Thermo Fisher Scientific (2 mentions) Axio imager z2 microscope, by Zeiss (2 mentions) Dm4000 fluorescence microscope, by Leica camera (1 mentions) Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions) Iba 1, by Abcam (1 mentions) Cresyl violet, by Merck Group (1 mentions) Lm3050s, by Leica camera (1 mentions) Glial fibrillary acidic protein (gfap), by Abcam (1 mentions) Il 1β, by Abcam (1 mentions) Image pro plus software, by Olympus (1 mentions) Leica dmi8, by Leica Microsystems (1 mentions)

4 protocols using vectashield antifade with dapi

Phagocytosis of M. bovis BCG by THP-1 cells

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THP-1 cells were cultured as described above and seeded at 1 × 10⁵ cells per 13 mm coverslip and differentiated with PMA as described above. GFP-expressing M. bovis BCG was incubated with 0, 1, 10 µg/ml of TSR4+5 for 2 h at 37°C in buffer 1. Cells were also incubated with 10 µg/ml of BSA as a negative control. Cells were washed twice in PBS and then resuspended in plain RPMI media. 1 × 10⁶ GFP-M. bovis BCG was added to the THP-1 cells (MOI of 10:1) and incubated for phagocytosis for 2 h at 37°C. THP-1 cells were then washed three times in PBS to remove extracellular bacteria and then fixed in 4% paraformaldehyde for 5 min. After washing three times in PBS, THP-1 cells were incubated with 2 µg/ml of AlexaFluor546-conjugated wheat germ agglutinin (Invitrogen) to reveal the plasma membrane. Cells were then washed three times and mounted using Vectashield antifade with DAPI (Vector Labs) to reveal nucleus. Slides were observed under a Leica DM4000 fluorescence microscope at 40× magnification. Images were processed using Image J (see text footnote 1).

Al-Mozaini M.A., Tsolaki A.G., Abdul-Aziz M., Abozaid S.M., Al-Ahdal M.N., Pathan A.A., Murugaiah V., Makarov E.M., Kaur A., Sim R.B., Kishore U, & Kouser L. (2018). Human Properdin Modulates Macrophage: Mycobacterium bovis BCG Interaction via Thrombospondin Repeats 4 and 5. Frontiers in Immunology, 9, 533.

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Zebrafish Sperm Preparation and Imaging

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To collect wild-type or mutant sperm, male zebrafish were anesthetized using 0.1% tricaine. Sperm was collected with a glass capillary from the urogenital opening and immediately fixed with 3.7% formaldehyde at 4°C for 20 min. Sperm were spun onto an adhesive slide using a CytoSpin 4 (Thermo Fisher Scientific) at 1,000 rpm for 5 min followed by permeabilization with ice-cold methanol for 5 min and a wash with 0.1% Tween in 1x PBS (PBST). After mounting using VECTASHIELD Antifade with DAPI (Vector Laboratories), sperm were imaged with an Axio Imager.Z2 microscope (Zeiss) with an oil immersion 63x/1.4 Plan-Apochromat DIC objective.

Binner M.I., Kogan A., Panser K., Schleiffer A., Deneke V.E, & Pauli A. (2022). The Sperm Protein Spaca6 is Essential for Fertilization in Zebrafish. Frontiers in Cell and Developmental Biology, 9, 806982.

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Immunofluorescent Labeling of Zebrafish Sperm

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Sperm from zebrafish males was collected in 3.7% formaldehyde diluted in Hank’s saline solution and stored on ice for 20 min to 1 h. Sperm was pelleted by centrifugation at 850 rpm for 3 min, and the fixative was replaced with Hank’s saline. Sperm was spun onto an adhesive slide with a CytoSpin 4 (Thermo Fisher Scientific) at 800 rpm for 3 min. Slides were briefly washed once in 1x PBS, and the sperm was permeabilized in 0.25% Tween in 1x PBS for 30 min before blocking with 10% normal goat serum (Invitrogen) and 40 μg/ml BSA in PBST for at least 1 h. Slides were then incubated with mouse anti-zebrafish-Dcst2 antibody in blocking buffer (1:650; (Noda et al., 2021 )) overnight at 4°C in a humidified chamber. After several washes with PBST, slides were incubated with goat anti-mouse IgG Alexa Fluor 488 secondary antibody (1:380, Thermo Fisher Scientific) for 1 h, washed several times with PBST and finally once with 1x PBS. After mounting using VECTASHIELD Antifade with DAPI (Vector Laboratories), sperm was imaged with an Axio Imager.Z2 microscope (Zeiss) using an oil immersion 100x/1.4 plan-apochromat objective. Widefield sperm images were processed for each genotype using Fiji by adjusting image brightness and contrast without clipping of intensity values.

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Neuroinflammatory Changes After Hypoxic-Ischemic Injury

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Rats were anesthetized and perfused with cold PBS followed by 4% formalin at 24 h post HI. The brains were taken out and post-fixed overnight, then immersed in 30% sucrose until dehydrated and frozen in OCT. Coronal sections were cut at 10 μm thickness using a cryostat (Leica LM3050S). For nissl staining the slides were dehydrated in 95% and 70% ethanol for 1 min respectively, stained with 0.5% cresyl violet (Sigma-Aldrich, USA) for 2 min, and then dehydrated in 100% ethanol and xylene for 1.5 min consecutively. The percentage of brain tissue loss was calculated with the same equation for infarct area.
Immunofluorescent staining was performed as described previously (Ye et al., 2018 (link)). Briefly, after being permeabilized with 0.3% Triton X-100 and blocked by 5% donkey serum, sections were incubated with primary antibodies (FPR2, 1:100, Abcam, USA; Iba1, 1:100, Abcam, USA; GFAP, 1:100, Abcam, USA; IL-1β, 1:200, Abcam, USA; MPO, 1:200, Abcam, USA) at 4°C overnight. The next day sections were incubated with appropriate fluorescent dye-conjugated secondary antibodies (1:200) for 2 h at room temperature and mounted using Vectashield Antifade with DAPI (Vector Laboratories Inc., USA). The stained sections were captured under a fluorescence microscope Leica DMi8 (Leica Microsystems, Germany) and analyzed with Image Pro Plus software (Olympus, Melville, NY).

Liu W., Huang J., Doycheva D., Gamdzyk M., Tang J, & Zhang J.H. (2019). RvD1binding with FPR2 attenuates inflammation via Rac1/NOX2 pathway after neonatal hypoxic-ischemic injury in rats. Experimental neurology, 320, 112982.

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