Vectashield solution containing dapi

Manufactured by Vector Laboratories

Vectashield solution containing DAPI is a mounting medium used for fluorescence microscopy. It is designed to protect fluorescent signals and provide nuclear counterstaining with the fluorescent dye DAPI.

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Lab products found in correlation

Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions) Metamorph, by Molecular Devices (1 mentions) Huygens professional, by Scientific Volume Imaging (1 mentions) 2200 analyzer, by INCELL (1 mentions) 510 meta, by Zeiss (1 mentions) Bovine serum albumin fraction 5, by Merck Group (1 mentions) Triton x 100, by Merck Group (1 mentions) Oct3 4, by Santa Cruz Biotechnology (1 mentions) Alexa fluor 488 or 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions) Tandem confocal microscope, by Leica (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) γ tubulin, by Merck Group (1 mentions) Bodipy fl, by Thermo Fisher Scientific (1 mentions) Hvin 1, by Merck Group (1 mentions) Cy3 conjugated goat anti mouse or anti rabbit, by Jackson ImmunoResearch (1 mentions) Anti fibronectin rabbit polyclonal, by Merck Group (1 mentions)

6 protocols using vectashield solution containing dapi

Immunofluorescence and RNA FISH Protocol

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IF experiments were carried out on cells grown on coverslips. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Coverslips were incubated with primary antibodies in 3% BSA for 1 h at room temperature. Coverslips were washed three times with PBS and incubated with secondary conjugated antibodies for 45 min. Coverslips were wash 3 times in PBS and fixed again before doing RNA FISH experiments. SmiFISH method was done as previously described (44 (link)) using Cy3-labeled DNA fragments specific to U3 and U85 snoRNAs. After incubation with the hybridization mix for one night, coverslips were all washed twice in 10% formamide in 2XSSC, once in PBS and mounted in Vectashield solution containing DAPI (Vector Laboratories). Samples were observed at RT using an upright epifluorescence microscope (LEICA DM6000) with a ×63 oil objective (NA 1.3). Images were captured with a CCD camera (Coolsnap HQ2 from Photometrics) using MetaMorph (Molecular Devices) and processed with Photoshop (Adobe). Deconvolution was proceeded with Huygens Professional (Scientific Volume Imaging).

Abel Y., Paiva A.C., Bizarro J., Chagot M.E., Santo P.E., Robert M.C., Quinternet M., Vandermoere F., Sousa P.M., Fort P., Charpentier B., Manival X., Bandeiras T.M., Bertrand E, & Verheggen C. (2020). NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis. Nucleic Acids Research, 49(2), 1094-1113.

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Immunofluorescence Assay for Endosomal Markers

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Fibroblasts were cultured in chamber slides for 72 hrs after passage and washed with PBS twice, fixed with 4% PFA at room temperature for 15 min, and permeabilized using 0.1% saponin. Slides were blocked with 5% donkey serum and 1% BSA for 30 min, incubated in primary Abs overnight at 4°C followed by incubation in appropriate secondary Abs, mounted in Vectashield solution containing DAPI (Vector Laboratories). Antibodies used for this method were: rabbit polyclonal anti-EEA1 Ab (Abcam), rabbit polyclonal anti-Rab11 Ab (Abcam), rabbit monoclonal anti-Rab7 AB (Cell Signaling), mouse monoclonal anti-CD63/LAMP3 Ab (H5C6; Developmental Studies, Hybridoma Bank), and Alexa fluor secondary donkey anti-rabbit/mouse Abs (Invitrogen). Cells were imaged with a Zeiss 510 META confocal laser-scanning microscope. For quantification of CD63 staining, 6000 cells/well were seeded in 96-well black/clear bottom plates and stained with anti-CD63/LAMP3 Ab, followed by donkey anti-mouse Alexa fluor secondary antibody. Cells were then imaged on the InCell 2200 Analyzer and quantified as described elsewhere [11 (link)].

Stephen J., Yokoyama T., Tolman N.J., O’Brien K.J., Nicoli E.R., Brooks B.P., Huryn L., Titus S.A., Adams D.R., Chen D., Gahl W.A., Gochuico B.R, & Malicdan M.C. (2017). Cellular and molecular defects in a patient with Hermansky-Pudlak syndrome type 5. PLoS ONE, 12(3), e0173682.

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Immunohistochemical Labeling of Utricles

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The utricles were placed in a rotary shaker and incubated for 3 hours in a blocking solution containing 2% bovine serum albumin fraction V (Sigma, SLM), 0.1% Triton X-100 (Sigma, SLM) diluted in PBS at 4°C to 6°C. Subsequently, the blocking solution is removed, and the whole organs are incubated for 72 hours with the primary antibodies, placing the vials in the rotatory shaker in a cold room. At the end of the incubation, the secondary antibodies against rabbit labeled with Alexa 488 and against mouse labeled with Alexa 594 were diluted 1:1000 in PBS (Molecular Probes), applied to the tissue sections, and incubated for 1 hour at room temperature in the dark. At the end of the incubation, the whole endorgans or sections were washed with PBS (20 minutes × 5) and mounted flat on glass slides with Vectashield solution containing DAPI (Vector Labs).

Lopez I.A., Ishiyama G., Acuna D, & Ishiyama A. (2019). Otopetrin-2 Immunolocalization in the Human Macula Utricle. The Annals of otology, rhinology, and laryngology, 128(6 Suppl), 96S-102S.

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Immunofluorescence Characterization of Stem Cells

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At the time points indicated, cultured cells were fixed with 4% paraformaldehye and blocked in 20% normal goat serum. Cells were incubated with primary antibody [Oct-3/4, PDGFRα and Nanog (Santa Cruz biotechnology, 1:200 dilution), Nestin and NG2 (Millipore, 1:200 dilution), Pax6 (Covance, 1:200 dilution)] at 4 °C for overnight then washed three times with PBS. Cells were subsequently incubated with goat anti-mouse or anti-rabbit Alexafluor-488 or 594 conjugated secondary antibody [(1:1000 dilution), Invitrogen] for 1 h at room temperature and washed five times with PBS. Stained cells were washed once with H₂O, air dried and then mounted with Vectashield solution containing DAPI (Vectorlabs). Staining was analyzed by using Nikon Eclipse 2000 inverted fluorescence microscope. Lineage marker expression values were quantified as the percentage of total DAPI⁺ cells in each culture field of view that expressed the lineage-specific marker of interest. Data are expressed as the mean percent positive cells±SEM from at least 5 random fields of view from at least 3 independent cultures.

Kim S.Y., Kelland E.E., Kim J.H., Lund B.T., Chang X., Wang K, & Weiner L.P. (2016). The influence of retinoic acid on the human oligodendrocyte precursor cells by RNA-sequencing. Biochemistry and Biophysics Reports, 9, 166-172.

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Immunofluorescence Staining for SIK2 and γ-Tubulin

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Cells were seeded at a density of 50,000 cells per well in a 24-well plate with coverslips. After at least 48 h to allow adherence, cells were washed in 1× PBS and were fixed with 4 % paraformaldehyde (Sigma-Aldrich) for 3 min. Cells were then washed in cold 1× PBS (Gibco), treated with 80 % ice cold ethanol and stored at −20°C until staining. For staining, cells were washed in 1× TBS-0.2 % Triton-0.04 % SDS, blocked for 30 min with 1× TBS supplemented with 1.5 % bovine serum albumin (BSA) (Sigma-Aldrich) and incubated for 1 h with a SIK2 (BioLegend) or γ-Tubulin (Sigma-Aldrich) primary antibody followed by an appropriate AlexaFluor488® or AlexaFluor594® conjugated secondary antibody (Invitrogen). Cover slips were then mounted onto slides with Vectashield solution containing DAPI (Vectashield). Images were taken using a Leica Tandem confocal microscope.

Bon H., Wadhwa K., Schreiner A., Osborne M., Carroll T., Ramos-Montoya A., Ross-Adams H., Visser M., Hoffmann R., Ahmed A.A., Neal D.E, & Mills I.G. (2014). Salt-inducible Kinase 2 Regulates Mitotic Progression and Transcription in Prostate Cancer. Molecular cancer research : MCR, 13(4), 620-635.

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Immunofluorescence Analysis of Cytoskeletal Proteins

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After 4 hours of culture, the culture medium was removed and unattached cells were washed away from the surface with PBS. Cells were fixed with 3 % paraformaldehyde in PBS for 30 min at 4 ⁰C, and washed three times with PBS. After permeabilizing with 0.1% Triton-X in PBS for 5 min at RT and blocking with DPBS/BSA 1 % at RT for 30 min, samples were incubated with a primary antibody for 1 hour at RT; the antibody used to analyze vinculin expression was antivinculin (mouse) (hVIN-1, Sigma-Aldrich) 1:400 in DPBS/BSA 1% and to analyze fibronectin expression was anti-fibronectin (rabbit) (polyclonal, Sigma-Aldrich) 1:400 in DPBS/BSA 1%.
Samples were washed twice with DPBS/Tween 20. A combination of secondary antibody (Cy3conjugated goat anti-mouse or antirabbit, respectively, Jackson ImmunoResearch) and phalloidin
(1:100) (BODIPY FL, Life Technology) was added and incubated by 1 hour at RT. Finally, after washing the samples with DPBS/Tween 20 three times, a mounting with Vectashield solution containing DAPI (Vector Laboratories) was performed.

Marín-Pareja N., Cantini M., González-García C., Salvagni E., Salmerón-Sánchez M, & Ginebra M.P. (2015). Different Organization of Type I Collagen Immobilized on Silanized and Nonsilanized Titanium Surfaces Affects Fibroblast Adhesion and Fibronectin Secretion. ACS applied materials & interfaces, 7(37).

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