Vectashield dapi solution

Manufactured by Vector Laboratories

Sourced in United States

Vectashield/DAPI solution is a fluorescent mounting medium designed for preserving fluorescent signals in microscopy samples. The solution contains the fluorescent dye DAPI, which binds to DNA and emits blue fluorescence. The Vectashield component helps to maintain the brightness and stability of fluorescent signals.

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Lab products found in correlation

Eclipse ts100 microscope, by Nikon (2 mentions) Ts2 fl microscope, by Nikon (1 mentions) Ds fi2 camera, by Nikon (1 mentions) Ds fi2 epifluorescence microscope, by Olympus (1 mentions) Leica sp2 aobs confocal microscope, by Leica camera (1 mentions) Af594, by Thermo Fisher Scientific (1 mentions) Poly l lysine (pll), by Merck Group (1 mentions) Triton x 100, by Merck Group (1 mentions) Axioplan 2 fluorescent microscope, by Zeiss (1 mentions) 24 well plate, by BD (1 mentions) Jbw301, by Merck Group (1 mentions) Erythrocyte lysis buffer, by Qiagen (1 mentions)

8 protocols using vectashield dapi solution

Immunofluorescence Analysis of Cas9-Mediated Genetic Manipulation

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Cas9 expressing RH4 cells were transduced with indicated sgRNA as described above. Cells were plated onto chamber slides and immunofluorescence was performed after 7 days post transduction. For experiments with BirA constructs, immunofluorescence was carried out the day after transfection as described above. After washing with 1xPBS, cells were fixed with 4% Formalin followed by washing and quenching with 0.1 M Glycine/PBS. Cells were washed three more times with 1xPBS and permeabilized with 0.1% Triton X-100/PBS and blocked using 4% horse serum in 0.1% Triton X-100/PBS. Incubation with primary antibody dissolved in 4% horse serum in 0.1% Triton X-100/PBS was done overnight in a humid chamber. The next day, secondary antibodies were added in 4% horse serum/PBS for 1 h. Antibodies used for Immunofluorescence are indicated in Supplementary Data S5B. After washing three times with 1xPBS, slides were embedded and counterstained with Vectashield/DAPI solution (Vector Laboratories, H-1200) and sealed with nail polish.

Laubscher D., Gryder B.E., Sunkel B.D., Andresson T., Wachtel M., Das S., Roschitzki B., Wolski W., Wu X.S., Chou H.C., Song Y.K., Wang C., Wei J.S., Wang M., Wen X., Ngo Q.A., Marques J.G., Vakoc C.R., Schäfer B.W., Stanton B.Z, & Khan J. (2021). BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma. Nature Communications, 12, 6924.

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Immunofluorescent Staining of Tight Junctions

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For staining the tight junction protein ZO‐1, HUVECs were seeded at a density of 300 000 cell·well⁻¹ in 6‐well plates. After 24 h of AAV2 infection, HUVECs were synchronized with EBM™‐2 supplemented with 2% FBS for 24 h. After 30 min of VEGF treatment, HUVECs were fixed with acetone:methanol (1 : 1) on ice for 10 min. After washing with PBS and blocking with 1% BSA in PBST (0.1% Tween 20) for 30 min, HUVECs were incubated with the anti‐ZO‐1 antibody at 4 °C for 16 h. The cells were again washed with PBS and incubated with anti‐rabbit IgG‐FITC for 1 h. Then, HUVECs were mounted with the VECTASHIELD® DAPI solution (Vector Laboratories, CA, USA), and images were obtained using a Nikon Ts2 FL Microscope (Nikon, Tokyo, Japan).

Cha S., Seo W., Woo H., Kim H.J., Lee S.H., Kim J., Choi J., Park K., Lee J.Y., Lee B.J, & Lee H. (2021). AAV expressing an mTOR‐inhibiting siRNA exhibits therapeutic potential in retinal vascular disorders by preserving endothelial integrity. FEBS Open Bio, 12(1), 71-81.

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Paraformaldehyde-based Cell Fixation and Fluorescence Imaging

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Cells were fixed using 4% paraformaldehyde for 10 min at room temperature. After fixation, three washes with PBS 1× solution were performed, and a final wash in distilled water was made to remove excess salt. Finally, the coverslips were mounted with a Vectashield/DAPI solution (Vector). Images were acquired in dark and light conditions with a DS-Fi2 epifluorescence microscope (Olympus, Shinjuku-ku, Tokyo, Japan) equipped with a Nikon DS-Fi2 camera (Nikon, Minato-ku, Tokyo, Japan) operated with QCapture Suite PLUS 3.1.3.10 (Q-Imaging).

Arancibia D., Pol I., Vargas-Fernández M., Zárate R.V., Signorelli J.R, & Zamorano P. (2023). OPTO-BLUE: An Integrated Bidirectional Optogenetic Lentiviral Platform for Controlled Light-Induced Gene Expression. International Journal of Molecular Sciences, 24(11), 9537.

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Immunolocalization of TCR Beta Subunit

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Cells were seeded in circular cover glasses, preincubated with 0.005% poly-L-lysine (Sigma-Aldrich) for 30 min at 37°C, and rinsed several times. Cells were then fixed with 4% p-formaldehyde in PBS for 5 min and, after rinsing, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS (both steps at room temperature, 20–22°C). Nonspecific binding sites were blocked by incubation for 1 h in PBS containing 10 μg/ml human IgG and a commercial blocking reagent (Roche, Basel, Switzerland). Incubation with primary TCRβ antibody (JOVI-1) or secondary anti-mouse antibody AF594 (Invitrogen) was done for 1 h or 30 min at 20–22°C, respectively. Appropriate isotype-matched IgG or normal serum was used in parallel as the control. Stained cells were mounted in Vectashield^® -DAPI solution (Vector Laboratories, Burlingame, CA, United States) and visualized under a Leica SP-2 AOBS confocal microscope (HCX PL APO × 63 1.4 oil-immersion objective; 1.400,000, numerical aperture) and TCSNTV software (Leica Microsystems, Wetzlar, Germany). Images were software processed with ImageJ.

Garcillán B., Fuentes P., Marin A.V., Megino R.F., Chacon-Arguedas D., Mazariegos M.S., Jiménez-Reinoso A., Muñoz-Ruiz M., Laborda R.G., Cárdenas P.P., Fernández-Malavé E., Toribio M.L, & Regueiro J.R. (2021). CD3G or CD3D Knockdown in Mature, but Not Immature, T Lymphocytes Similarly Cripples the Human TCRαβ Complex. Frontiers in Cell and Developmental Biology, 9, 608490.

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Immunofluorescence Analysis of Cas9-Edited Cells

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Cas9 expressing RH4 cells were transduced with indicated sgRNA as described above. Cells were plated onto chamber slides and immunofluorescence was performed after 7 days post transduction. For experiments with BirA constructs, immunofluorescence was carried out the day after transfection as described above. After washing with 1xPBS, cells were fixed with 4% Formalin followed by washing and quenching with 0.1M Glycine/PBS. Cells were washed 3 more times with 1xPBS and permeabilized with 0.1% Triton X-100/PBS and blocked using 4% horse serum in 0.1% Triton X-100/PBS. Incubation with primary antibody dissolved in 4% horse serum in 0.1% Triton X-100/PBS was done overnight in a humid chamber. The next day, secondary antibodies were added in 4% horse serum/PBS for 1h. Antibodies used for Immunofluorescence are indicated in Table S5B. After washing 3 times with 1xPBS, slides were embedded and counterstained with Vectashield/DAPI solution (Vector Laboratories, H-1200) and sealed with nail polish.

Laubscher D., Gryder B.E., Sunkel B.D., Andrésson Þ., Das S., Roschitzki B., Wolski W., Wu X., Chou H., Song Y., Wang C., Wei J.S., Wang M., Wen X., Marques J.G., Wachtel M., Vakoc C.R., Schäfer B.W., Stanton B.Z., & Khan J. (2021). BAF complexes drive proliferation and block myogenic differentiation in fusion-positive rhabdomyosarcoma.

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Quantifying Tumor Cell Invasion

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Tumor cell invasion assays were performed using modified Boyden chambers consisting of 8 μm pore filter inserts in 24-well plates (BD Biosciences, San Jose, CA) as described elsewhere [35 (link)]. PC-3 cells were trypsinized and resuspended in serum-free RPMI-1640 medium and plated on Matrigel-coated membranes of the upper compartments. Cells were incubated at 37°C for 22 hours in a CO₂ incubator, using 5% fetal bovine serum in the lower chambers as a chemoattractant. Following incubation, the inserts were pulled out and the non-invading cells on the upper surface were removed with a cotton swab. The cells on the lower surface of the membrane were fixed in 4% paraformaldehyde, air-dried and stained with DAPI VECTASHIELD solution (Vector Laboratories Inc., Burlingame, CA). Membranes were scanned using a Zeiss AxioPlan 2 fluorescent microscope. The number of invaded cells was counted. Results were expressed as means ± SD. Statistical significance was established using the Student's t-test.

Lin D., Dong X., Wang K., Wyatt A.W., Crea F., Xue H., Wang Y., Wu R., Bell R.H., Haegert A., Brahmbhatt S., Hurtado-Coll A., Gout P.W., Fazli L., Gleave M.E., Collins C.C, & Wang Y. (2014). Identification of DEK as a potential therapeutic target for neuroendocrine prostate cancer. Oncotarget, 6(3), 1806-1820.

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Quantifying DNA Double-Strand Breaks

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γ-H2AX foci, indicative of DSBs (Rogakou et al. 1999 (link)), were determined in peripheral blood mononuclear cells by immunofluorescent assay as in our previous studies (Kovvuru et al. 2015 (link); Nallanthighal et al. 2017 (link)). Briefly, 50 µl of peripheral blood was incubated with 10× (v/v) erythrocyte lysis buffer (8.5 g/l ammonium chloride in 0.01 M Tris-HCl buffer; pH 7.5) to lyse erythrocytes. The remaining cells were suspended in 50 µl of PBS, pipetted onto poly-d-lysine-coated cover slips, fixed with 4% of paraformaldehyde, permeabilized with 0.5% of Triton-X 100 and washed with PBS. After blocking, cells were incubated with mouse anti-phospho-Histone2AX (Ser139) antibody clone JBW301 (EMD Millipore, Billerica, MA, Catalog #05-636) at a 1:400 dilution followed by incubation with FITC-conjugated anti-mouse IgG secondary antibody (Jackson Immunochemicals, West Grove, PA, Catalog #115-095-008) at a 1:200 dilution. Coverslips were mounted on microscopic slides with 7 µl of DAPI/Vectashield solution (Vector Laboratories, Burlingame, CA) to counterstain nuclei. Slides were visualized under a 100× objective on a Nikon Eclipse TS100 microscope. A minimum of 100 cells were analyzed and cells with more than four distinct foci in the nucleus were considered positive for γ-H2AX foci (Figure 1(B)). Data were analyzed blinded to treatment.

Nallanthighal S., Chan C., Murray T.M., Mosier A.P., Cady N.C, & Reliene R. (2017). Differential effects of silver nanoparticles on DNA damage and DNA repair gene expression in Ogg1-deficient and wild type mice. Nanotoxicology, 11(8), 996-1011.

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Quantification of DNA Damage Foci

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Fifty μl of peripheral blood was incubated with erythrocyte lysis buffer (Qiagen, Germantown, MD) to eliminate red blood cells. Cells were suspended in 50 μl of PBS and applied onto poly-D-lysine coated cover slips. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton-X 100 and washed with PBS as described previously (37 (link)). After blocking, cells were incubated with mouse anti-phospho-Histone2AX (Ser139) antibody clone JBW301 (EMD Millipore, Billerica, MA, Catalog #05-636) at a 1:400 dilution followed by incubation with FITC-conjugated anti-mouse IgG secondary antibody (Jackson Immunochemicals, West Grove, PA, Catalog #115-095-008) at a 1:200 dilution. Coverslips were mounted on microscopic slides with 5 μl of DAPI/Vectashield solution (Vector Laboratories, Burlingame, CA). Slides were visualized under a 100X objective on a Nikon Eclipse TS100 microscope. A minimum of 100 cells were analyzed and cells with more than four distinct foci in the nucleus were considered positive for γ-H2AX foci formation.

Nallanthighal S., Shirode A.B., Judd J.A, & Reliene R. (2016). Pomegranate intake protects against genomic instability induced by medical x-rays in vivo in mice. Nutrition and cancer, 68(8), 1349-1356.

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