Vectashield with dapi

Manufactured by Vector Laboratories

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Vectashield with DAPI is a mountant medium designed for fluorescence microscopy. It contains the fluorescent dye DAPI, which selectively binds to DNA and emits blue fluorescence when exposed to ultraviolet light. This product is used to mount and preserve fluorescently-labeled samples for microscopic analysis.

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Lab products found in correlation

Alexa fluor 488, by Thermo Fisher Scientific (24 mentions) Triton x 100, by Merck Group (17 mentions) Alexa 488, by Thermo Fisher Scientific (17 mentions) Alexa fluor 594, by Thermo Fisher Scientific (16 mentions) Paraformaldehyde (pfa), by Merck Group (12 mentions) Bovine serum albumin (bsa), by Merck Group (12 mentions) Sp5 confocal microscope, by Leica (11 mentions) Rhodamine phalloidin, by Thermo Fisher Scientific (11 mentions) Lsm 700, by Zeiss (10 mentions) Tcs sp8 confocal microscope, by Leica (9 mentions) Lsm 700 confocal microscope, by Zeiss (8 mentions) Vt1000s vibratome, by Leica (8 mentions) Lsm 710 confocal microscope, by Zeiss (8 mentions) Axiovision software, by Zeiss (8 mentions) Sp8 confocal microscope, by Leica (7 mentions) Alexa 555, by Thermo Fisher Scientific (7 mentions) Lsm 780 confocal microscope, by Zeiss (6 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (6 mentions) Normal goat serum (ngs), by Jackson ImmunoResearch (6 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (6 mentions)

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738 protocols using vectashield with dapi

Visualizing Drosophila Fat Bodies and Hemocytes

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Fat body dissections were performed on flies expressing GFP-mCherry-Atg8a. Flies were cut transversally with a scalpel on a petri dish cleaned with 70% ethanol to observe the fat bodies. Fat bodies were fixed with PFA 8% and mounted on diagnostic microscope slides (Thermo Fisher Scientific) in Vectashield with DAPI (Vector Laboratories).
To observe hemocytes, wild-type larvae injected with untreated or heat killed spores (treatment at 100°C for 15 minutes) were opened 6 hours after injection in a drop of 1x PBS directly on diagnostic microscope slides. After dissection, samples were left for 30 minutes to settle the cells on the slides. Hemocytes were fixed with 8% PFA, permeabilized for 15 min with 1x PBS and 0.1% Triton X-100. Samples were blocked for 2h in 1x PBS, 0.1% Triton X-100 and 2% BSA (PTB). Hemocytes were incubated in PTB plus the primary rabbit antibody anti-Tr spores (1/500) and 10 μM of FITC phalloidin (Sigma-Aldrich #P5282). Samples were washed for 15 min with 1x PBS and 0.1% Triton X-100. Cells were incubated for 2h on PTB plus the secondary goat antibody anti-rabbit coupled to Cy3 (1/500) (Invitrogen #A10522). Hemocytes were washed for 15 min with 1x PBS and 0.1% Triton X-100 and mounted in Vectashield with DAPI (Vector Laboratories). All samples were observed using a LSM 780 confocal microscope (Zeiss).

Caravello G., Franchet A., Niehus S, & Ferrandon D. (2022). Phagocytosis Is the Sole Arm of Drosophila melanogaster Known Host Defenses That Provides Some Protection Against Microsporidia Infection. Frontiers in Immunology, 13, 858360.

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Immunofluorescence Staining of Cells and Chromosomes

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DT40 cells were fixed (4% paraformaldehyde in PBS), permeabilized (0.25% Triton-X 100, 1% BSA in PBS), washed and incubated with antibodies on Polysine-coated slides. Slides were mounted in Vectashield with DAPI (Vector laboratories). For immunofluorescence on chromosome spreads, human lymphocytes were treated with 0.1 μg ml⁻¹ Karyomax Colcemid (Gibco) for 4 h before being harvested and centrifuged onto glass slides using a Cytospin 4 (Thermo Scientific). The spreads were fixed, incubated with antibodies and mounted in Vectashield with DAPI (Vector laboratories). Alexa Fluor (Life technologies) fluorophore-conjugated secondary antibodies were used to visualize the primary antibodies. Images were captured on either a Nikon Ellipse microscope, an automated upright Olympus BX63 fluorescence microscope or an upright point scanning confocal Axioimager Z2 microscope (Zeiss). Images from confocal microscopy were analysed with ZEN 2010 (Zeiss) otherwise ImageJ was used for image analysis31 (link).

Nielsen C.F., Huttner D., Bizard A.H., Hirano S., Li T.N., Palmai-Pallag T., Bjerregaard V.A., Liu Y., Nigg E.A., Wang L.H, & Hickson I.D. (2015). PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis. Nature Communications, 6, 8962.

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Immunofluorescence Imaging of Kidney Tissue

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Kidneys were harvested from animals at 32 weeks, 52 weeks, or 8 weeks after chronic apoptotic thymocyte injection (above). Organs were sectioned and mounted on slides. Slides were fixed with 4% formaldehyde for 20 minutes at 4°C. Following fixation, slides were blocked and permeabilized in block buffer (1% BSA, 0.1% Triton in PBS) for 1 hour at RT. Slides were washed extensively in TBS-Tween (Tris-buffered saline containing 0.05% Tween-20), incubated with Alexa-Fluor 647-conjugated anti-IgG (Invitrogen) for 1 hour at RT, and mounted with VectaShield with DAPI (Vector Labs). Alternatively, slides were washed extensively in TBS-Tween (Tris-buffered saline containing 0.05% Tween-20), incubated with anti-C1q (clone 4.8, Abcam) for 1 hour at RT, washed again with TBS-Tween, incubated with Cy3 conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) and Alexa Fluor 488-conjugated wheat germ agglutinin (Molecular Probes) for 1 hour at RT, and mounted with VectaShield with DAPI (Vector Labs). Images were analyzed using an Olympus BX51 FL Microscope and Slidebook software. Masks were drawn around glomeruli, and MFI of anti-IgG or anti-C1q were calculated.

Martinez J., Cunha L.D., Park S., Yang M., Lu Q., Orchard R., Li Q.Z., Yan M., Janke L., Guy C., Linkermann A., Virgin H.W, & Green D.R. (2016). Noncanonical autophagy inhibits the auto-inflammatory, lupus-like response to dying cells. Nature, 533(7601), 115-119.

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Immunofluorescent Analysis of IL-33 and F4/80 in Colon Sections

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For immunofluorescent staining of IL-33 with F4/80 on colon sections, sections were deparaffinized, rehydrated, and antigen was unmasked in a high pH buffer (Vector Labs, Burlingame, CA). Sections were stained with goat anti-mouse IL-33 (AF3626, R&D Systems, Minneapolis, MN) followed by anti-goat-Alexa Fluor-594 (Life Technologies, Carlsbad, CA) with anti-F4/80-FITC (11-4801-11, eBioscience, San Diego, CA) and sections were counterstained with Vectashield with DAPI (Vector Laboratories, Burlingame, CA).
T84 cells grown on coverslips were subsequently fixed with 4% paraformaldehyde for 30 minutes. Cells were blocked with goat serum and then stained with rabbit anti-ST2 (ab25877, AbCam, Cambridge, MA) followed by goat anti-rabbit-Alexa Fluor488 (Jackson Immunoresearch, West Grove, PA), and Alexa Fluor635-Phalloidin (Life Technologies, Carlsbad, CA) and counterstained with Vectashield with DAPI (Vector Laboratories, Burlingame, CA).

Waddell A., Vallance J.E., Moore P.D., Hummel A.T., Wu D., Shanmukhappa S.K., Fei L., Washington M.K., Minar P., Coburn L.A., Nakae S., Wilson K.T., Denson L.A., Hogan S.P, & Rosen M.J. (2015). IL-33 signaling protects from murine oxazolone colitis by supporting intestinal epithelial function. Inflammatory bowel diseases, 21(12), 2737-2746.

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Immunofluorescence Analyses of Stem Cell Markers

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Individual GSCs were cystospun onto Thermo Shandon coated slides using Rotofix 32a (Hettich) centrifuge at 800 RPM for 5 minutes. Cells were fixed with 4% paraformaldehyde and incubated in blocking buffer for 1 hour at room temperature, followed by incubation in biotin conjugated anti-CD133 (Miltenyi Biotec) overnight at 4° C. Secondary antibody dilution was for 1 hour at room temperature using a Strepavidin conjugated Alexa Fluor antibody (Thermo Fisher Scientific). Slides were mounted using Vectashield with DAPI (Vector Labs), and imaged under 63X (oil immersion) using a Zeiss Axio Observer inverted fluorescent microscope.
T4213 neurospheres were plated in single wells of a poly-D-lysine culture slide. Culture slides were then incubated overnight in a humidified environment at 37°C and 5% CO₂. Slides were fixed with 4% paraformaldehyde, and then incubated overnight at 4°C with primary antibodies against SOX2 (Cell Signaling Technology) and Nestin (Abcam).

Sheikh S., Saxena D., Tian X., Amirshaghaghi A., Tsourkas A., Brem S, & Dorsey J.F. (2019). An Integrated Stress Response Agent that Modulates DR5-Dependent TRAIL Synergy Reduces Patient-Derived Glioma Stem Cell Viability. Molecular cancer research : MCR, 17(5), 1102-1114.

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Isolation of Adult Mouse Ventricular Cardiomyocytes

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Adult mouse ventricular cardiomyocytes were isolated using Langendorff perfusion as we described previously (Zhang & Nam, 2018 (link)). Dissected hearts were cannulated through the aorta and sequentially perfused with following buffers: (1) perfusion buffer (NaCl 120.4 mM, KCl 14.7 mM, Na₂HPO₄ 0.6 mM, Ka₂HPO₄ 0.6 mM, MgSO4 1.2 mM, Na-HEPES 10 mM, NaHCO₃ 4.6 mM, Taurine 30 mM, BDM 10 mM, Glucose 5.5 mM, pH 7.0), (2) digestion buffer without CaCl₂ (Collagenase II 2.4 mg/mL in perfusion buffer), and (3) digestion buffer with CaCl₂ (Collagenase II 2.4 mg/mL and CaCl₂ 40 μM in perfusion buffer). Perfused hearts were removed from the Langendorff apparatus and mechanically dissociated and triturated in stopping buffer (CaCl₂ 11.7 μM in calf serum 2 mL plus perfusion buffer 18 mL). Dissociated cells were centrifuged at low speed. The pellet, mainly composed of ventricular cardiomyocytes, was fixed with 4% PFA for 15 min. After PBS washing, a fraction of ventricular cardiomyocytes were immunostained in a 1.5 mL tube using a similar method described above. After immunostaining, a drop of cardiomyocyte containing solution was placed on glass slide and mounted with a coverslip and Vectashield with DAPI (VectorLabs). Images were captured using Olympus IX81 epifluorescent microscope.

Zhang Z., Zhang Q., Lal H, & Nam Y.J. (2019). Generation of Nppa-tagBFP reporter knock-in mouse line for studying cardiac chamber specification. Genesis (New York, N.Y. : 2000), 57(6), e23294.

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Transfection and Visualization of Myc-Rac1 in 3T3 Cells

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Swiss 3T3 cells were plated (5000 cells/well in 12-well plates) on coverslips previously coated with fibronectin (2.5 μg/ml) for 30 min at room temperature. The next day, Myc-tagged Rac1 constructs were transiently transfected into the cells using TransIT (Mirus) according to the manufacturer’s instructions. Twenty-four hours after transfection, the cells were fixed and stained with phalloidin and for the Myc-tag, as previously described [55 (link)]. Briefly, the cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences; Hatfield, PA, USA) overnight at 4 °C, permeabilized in 0.2% Triton X-100 (Sigma) in PBS for 5 min, and incubated with anti-Myc 911B antibody (1:500, Cell Signaling) for 1 h, followed by secondary AlexaFluor 488 anti-mouse antibody (1:500, Invitrogen) for 2 h and AlexaFluor 568 phalloidin (Invitrogen, 1:40 in PBS) for 30 min. The cells were incubated in the dark and rinsed in PBS three to five times between each step. Coverslips were mounted using 6 μl Vectashield with DAPI (Vector Laboratories; Burlingame, CA, USA). Cells were visualized and counted blindly for lamellipodia using a Nikon Eclipse TS100 microscope with a 40× objective. Representative images were recorded using a Zeiss LSM 710 confocal laser-scanning microscope with a 63× oil objective.

Hobbs G.A., Mitchell L.E., Arrington M.E., Gunawardena H.P., DeCristo M.J., Loeser R.F., Chen X., Cox A.D, & Campbell S.L. (2014). Redox regulation of Rac1 by thiol oxidation. Free radical biology & medicine, 79, 237-250.

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Cell Invasion Assay Optimization

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50,000 HTR8/SVneo cells were seeded on 100 µl 10x diluted Matrigel (Corning) coated 8.0 μm cell culture inserts in the absence or presence of ELA peptide (Tocris), Apelin-13 (Sigma) and ML221 non-peptide APLNR antagonist (Sigma). When invasion assays were performed using siRNA-transfected cells (as described above), from the harvested transfected cells 50,000 cells were seeded on 100 µl 16x diluted Matrigel (Corning) coated 8.0 μm cell culture inserts in the absence or presence of ELA peptide (Tocris) or Apelin-13 (Sigma). Invasion took place for 48 hours after which the membranes were fixed, mounted in Vectashield with DAPI (VectorLabs) and coverslipped, after which the cells on the underside of the membrane were counted. To count the cells pictures were taken of 9 random fields per membrane after which ImageJ was used to quantify the number of cells per picture.

Georgiadou D., Boussata S., Ranzijn W.H., Root L.E., Hillenius S., bij de Weg J.M., Abheiden C.N., de Boer M.A., de Vries J.I., Vrijkotte T.G., Lambalk C.B., Kuijper E.A., Afink G.B, & van Dijk M. (2019). Peptide hormone ELABELA enhances extravillous trophoblast differentiation, but placenta is not the major source of circulating ELABELA in pregnancy. Scientific Reports, 9, 19077.

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Immunofluorescent Detection of SDMA in FFPE Tissues

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Formalin-fixed, paraffin embedded tissues were sectioned, baked at 60°C for 30 min, dewaxed in xylene and rehydrated through an ethanol gradient per standard procedures, and then subject to antigen retrieval with citrate buffer (pH 6.0) in a pressure cooker. Tissues were washed in PBS-T (PBS +0.05% Triton X-100), blocked with 2% BSA in PBS-T, and then incubated overnight with 1:1000 anti-SDMA produced as previously described.⁴² (link) Tissues were washed, stained with anti-rabbit HRP (Cell Signaling), and Tyramide signal amplification (TSA) was performed with CF550R Dye (Biotium, #96077) in amplification buffer (Akoya Biosciences). Slides were subject to additional antigen retrieval in citrate buffer, and then the procedure repeated with anti-pan cytokeratin (clone AE1/3, Bethyl), anti-mouse HRP (Cell Signaling), and CF680R (Biotium, #92196). Finally, slides were mounted using Vectashield with DAPI (Vector Labs) and imaged using a Nikon Eclipse Ti2-e Fluorescence microscope. At least 5 images per tissue were captured and quantified using a custom MATLAB algorithm (R2020a, Mathworks). SDMA is reported as average SDMA intensity in the tissue after subtracting background intensity determined from a control with no primary antibody. All images per tissue were averaged to determine the SDMA intensity in that tissue.

Li Y., Dobrolecki L.E., Sallas C., Zhang X., Kerr T.D., Bisht D., Wang Y., Awasthi S., Kaundal B., Wu S., Peng W., Mendillo M.L., Lu Y., Jeter C.R., Peng G., Liu J., Westin S.N., Sood A.K., Lewis M.T., Das J., Yi S.S., Bedford M.T., McGrail D.J, & Sahni N. (2023). PRMT blockade induces defective DNA replication stress response and synergizes with PARP inhibition. Cell Reports Medicine, 4(12), 101326.

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Visualizing Exosome Uptake in Macrophages

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Exosomes were loaded with 1X CellMask Deep Red Plasma Membrane stain for 10 minutes at 37°C. Exosomes were then washed with PBS and centrifuged at 100,000 x g for 70 minutes in TLA 100.3 rotor (Beckman Coulter) to pellet exosomes and resuspended in culture medium.
For imaging, peritoneal macrophages (300,000 cells) were allowed to attach in eight-well chamber slides. Ten micrograms of exosomes loaded with CellMask were applied to peritoneal macrophages and imaged 24 hour following treatment. Exosome uptake was visualized by on a Leica TCS SP3 confocal microscope (Leica Microsystems Inc., Buffalo Grove IL). Cells were fixed in 2% paraformaldehyde and mounted with Vectashield with DAPI (Vector Laboratories Inc., Burlingame, CA) before imaging.

Rhein B.A., Powers L.S., Rogers K., Anantpadma M., Singh B.K., Sakurai Y., Bair T., Miller-Hunt C., Sinn P., Davey R.A., Monick M.M, & Maury W. (2015). Interferon-γ Inhibits Ebola Virus Infection. PLoS Pathogens, 11(11), e1005263.

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