Vectashield dapi

Manufactured by Vector Laboratories

Sourced in United States, United Kingdom, Germany

Vectashield/DAPI is a mounting medium used for fluorescence microscopy. It contains the fluorescent dye DAPI, which binds to DNA and emits blue fluorescence. Vectashield serves as a versatile antifade reagent that helps preserve the fluorescent signal during microscopic observation.

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Lab products found in correlation

Alexa fluor 488, by Thermo Fisher Scientific (8 mentions) Lsm 700 confocal microscope, by Zeiss (7 mentions) Triton x 100, by Merck Group (7 mentions) Bovine serum albumin (bsa), by Merck Group (7 mentions) Axio observer z1, by Zeiss (6 mentions) Confocal microscope, by Olympus (5 mentions) Lsm 780, by Zeiss (5 mentions) Lsm 780 confocal microscope, by Zeiss (5 mentions) Axio imager m1, by Zeiss (4 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (4 mentions) Lsm 710, by Zeiss (4 mentions) Axiophot microscope, by Zeiss (4 mentions) Axiovert 200m fluorescent microscope, by Zeiss (4 mentions) Tween 20, by Merck Group (4 mentions) Lsm 510 meta, by Zeiss (4 mentions) Alexa 488, by Thermo Fisher Scientific (4 mentions) Lsm 780 microscope, by Zeiss (3 mentions) Deadend fluorometric tunel system, by Promega (3 mentions) Nis elements software, by Nikon (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)

228 protocols using vectashield dapi

Anti-Nidogen Antibody Generation and Tissue Staining

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For generation of anti-Nidogen antibody, rabbits were immunized with epitope CTYVQEFDGERNADLIPC by Bio-med Biotechnology (Beijing, China). Embryos, fat bodies, wing imaginal discs and ovaries were stained using standard procedures and mounted in DAPI-Vectashield (Vector Laboratories, Burlingame, California). The following primary antibodies were used: rabbit anti-Ndg (1:2000, this study), chicken anti-betagalactosidase (1:500, AbCam, Cambridge, UK), chicken anti-GFP (1:500, AbCam), rabbit anti-Ndg (1:100, [34 (link)]). Secondary antibody is IgG conjugated to Alexa-555, IgG conjugated to Alexa-488 and Alexa 549 (1:200, Life technologies).
For lipid droplet staining, L3 larvae were turned inside out and fixed in 4% PFA for 20 minutes, washed twice in PBS and then incubated in a 1:1000 dilution in PBS of 1 mg/ml BODIPY 493/503 stock (Life Technologies) for 30 minutes, followed by two 10-min washes in PBS and mounting in DAPI-Vectashield (Vector Laboratories). Confocal images were obtained using a Leica (Wetzlar, Germany) SP2 microscope or a Zeiss (Oberkochen, Germany) LSM780 microscope equipped with a Plan-Apochromat 63X oil objective (NA 1.4). Eggs and pupae were imaged in a Leica M125 stereoscope. All images were processed with Adobe Photoshop and ImageJ.

Dai J., Estrada B., Jacobs S., Sánchez-Sánchez B.J., Tang J., Ma M., Magadán-Corpas P., Pastor-Pareja J.C, & Martín-Bermudo M.D. (2018). Dissection of Nidogen function in Drosophila reveals tissue-specific mechanisms of basement membrane assembly. PLoS Genetics, 14(9), e1007483.

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Immunofluorescence Detection of MUC5AC

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After hydration in PBS, the cells were blocked for 10 min in 2.5% bovine serum albumin and 0.2% Triton X-100 diluted in PBS, followed by PBS wash and 1-h incubation at room temperature in primary antibody diluted in 0.25% bovine serum albumin and 0.05% Tween 20. Then, the cells were rinsed three times in PBS and incubated for 1 h at room temperature with the secondary antibody goat anti-rabbit Alexa Fluor 488 (1:300, A11034, Life Technologies) diluted in 0.25% bovine serum albumin and 0.05% Tween 20. After rinsing three times in PBS, the cells were mounted with VectaShield-DAPI (Vector Laboratories) to counterstain nuclear DNA.
Immunofluorescence detection of MUC5AC on bulbar conjunctival imprints was performed as described previously [34 (link)]. Briefly, primary antibody against MUC5AC and the secondary antibody Alexa Fluor 488 (1:300, A11034, Life Technologies) were diluted in 0.1% bovine serum albumin; after staining, the cells were mounted with VectaShield-DAPI (Vector Laboratories).
Immunofluorescent images were acquired with an Olympus BX51 microscope and CCD-1300 camera (VDS Vosskühler GmbH, Osnabrück, Germany). The images were analyzed with NIS Elements software (Laboratory Imaging). The percentage of positive cells was counted on six randomly captured photographs per well (two wells per condition) from at least four independent tissue donors.

Stadnikova A., Trosan P., Skalicka P., Utheim T.P, & Jirsova K. (2019). Interleukin-13 maintains the stemness of conjunctival epithelial cell cultures prepared from human limbal explants. PLoS ONE, 14(2), e0211861.

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Comprehensive Antibody Staining Protocol

Cited 3 times

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The following primary antibodies were used: rabbit anti-Cg25C (Zang et al., 2015 (link); 1:5,000), rabbit anti-Ndg (Wolfstetter et al., 2009 (link); 1:2,000), guinea pig anti-Tango1 (Lerner et al., 2013 (link); 1:1,000), rabbit anti-Sec16 (Ivan et al., 2008 (link); 1:1,000), rabbit anti-GM130 (cat#ab30637, 1:500; Abcam), rabbit anti-COPII (Sec23, cat#PA1-069A, 1:500; Thermo Fisher Scientific), and goat anti-Gmap (Riedel et al., 2016 (link); 1:500). Secondary antibodies were IgG conjugated to Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 (1:200; Thermo Fisher Scientific). Larvae were predissected in PBS by turning them inside out, fixed in PBS containing 4% PFA, washed in PBS (3 × 10 min), blocked in PBT-BSA (PBS containing 0.1% Triton X-100 detergent, 1% BSA, and 250 mM NaCl), incubated overnight with primary antibody in PBT-BSA in 4°C, washed in PBT-BSA (3 × 20 min), incubated for 2 h with secondary antibody in PBT-BSA at room temperature, and washed in PBT-BSA (3 × 20 min) and PBS (3 × 10 min). Tissues were finally dissected and mounted on a slide with a drop of DAPI-Vectashield (cat#H-1200; Vector Laboratories). For blood cell staining and imaging, blood cells from two to five larvae were bled inside a 20-µl drop of PBS on a glass slide and allowed to attach to the slide for 10 min before fixation.

Liu M., Feng Z., Ke H., Liu Y., Sun T., Dai J., Cui W, & Pastor-Pareja J.C. (2017). Tango1 spatially organizes ER exit sites to control ER export. The Journal of Cell Biology, 216(4), 1035-1049.

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Immunostaining of Drosophila Testes

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All steps were performed at room temperature, unless described otherwise. All testes were dissected in PBT 0.2% Tween-20 and fixed for 20 minutes in 4% PFA. After washing twice with PBT, the testes were stored in methanol at -20°C. The testes were washed once with PBS, twice with PBT for 2x10 minutes, and “blocked” in PBT-10 for 1hr, before being incubated with primary antibody in PBT-1 (PBS + 1% BSA + 1% Tween-20) overnight with agitation at 4°C. After washing the testes 2x10 minutes with PBT-1, they were incubated with a 1:200 dilution of secondary antibody in PBT-1 for 2 hours. After 3x10 minutes washes in PBS, the samples were mounted in DAPI-Vectashield (Vector).
Antibodies: rabbit Vasa (1:5000, a gift from Prof. Paul Lasko), guinea pig Tj (1:10000, a gift from Dorothea Godt), chicken GFP (1:2000, Abcam), mouse FasIII (7G10) (1:100, DSHB), rabbit PH3 (1:500, Millipore). Secondary antibodies were Goat Anti-Chicken (DyLight 488, Abcam 96951), AlexaFluor568 goat anti-mouse (Molecular Probes A11004), AlexaFluor647 goat anti-rabbit (Molecular Probes A21245), and goat anti-guinea pig Cy3 (Novus Biologicals NB710-4179). DAPI in vectashield (Vector H-1200), was used for the visualization of DNA. All samples were examined under a 40x/1.3 or 63x/1.4–0.6 Oil DIC Plan-Neofluar objective, using a Leica LSM SP5 upright confocal microscope.

Francis D., Chanana B., Fernandez B., Gordon B., Mak T, & Palacios I.M. (2019). YAP/Yorkie in the germline modulates the age-related decline of germline stem cells and niche cells. PLoS ONE, 14(4), e0213327.

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Immunofluorescence analysis of RAGE and MAPK

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Cells were grown overnight on coverslips in complete medium at 37°C followed by serum starvation for 16 hours. PR3 (0.5 μg/mL) was added to the cells for 30 min at 37°C. After washes, cells were fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature (RT), permeabilized with 0.1% saponin, 0.1% BSA in TBS (30 min) and blocked with 1% BSA/TBS for 1 h at RT. Staining was performed with anti-RAGE antibody (Abcam) at 1 μg/mL, rabbit anti-PR3 antibody (Novus Biologicals) at 2 μg/mL, mouse monoclonal phospho-p44/42 (Thr202/Tyr204) E10 antibody (Cell Signaling) at 5 μg/mL, or rabbit anti-phospho-JNK (T183/Y185) antibody (R&D Systems) at 2 μg/mL. Secondary FITC-conjugated donkey anti-mouse antibody was used at 5 μg/mL and Cy3-conjugated goat anti-mouse or mouse anti-rabbit antibodies were used at 2.5 μg/mL (Jackson ImmunoResearch Laboratories). DNA was stained with Hoeschst 33258 (Sigma) or DAPI VectaShield (Vector Laboratories). Images were captured with an IX70 fluorescence microscope (Olympus) and confocal Leica TCS SP8.

Kolonin M.G., Sergeeva A., Staquicini D.I., Smith T.L., Tarleton C.A., Molldrem J.J., Sidman R.L., Marchiò S., Pasqualini R, & Arap W. (2017). Interaction between tumor cell surface receptor RAGE and proteinase 3 mediates prostate cancer metastasis to bone. Cancer research, 77(12), 3144-3150.

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Virus Infection Assay in Cell Lines

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HEK293-MOV10 or vector cells were plated in 8-well chamber slides (4×10⁴ cells/well). Cells were infected with VSV for up to 16h and fixed with paraformaldehyde (4% v/v), following permeabilization with Triton-X100 0.1%. Nuclei were stained with DAPI Vectashield (Vector Laboratories, Inc. Burlingame, CA). Human primary foreskin fibroblasts were plated in 8-well chamber slides (1×10⁴ cells/well) and transfected with siRNA using Lipofectamine RNAiMAX with either siRNA 10 nM or control siRNA for 72 h. Subsequently, cells were infected with EMCV for 16 h and fixed with paraformaldehyde (4% v/v), following permeabilization with Triton-X100 1%. Infected cells were stained using anti-dsRNA sera (J2) previously described (27 (link)) for 16 h at 4 °C. Immunofluorescence detection was carried out with conjugated anti-rabbit Alexa Fluor 488 and conjugated anti-mouse Alexa Fluor 594 (Invitrogen). Representative micrographs were obtained followed by quantitation of the % of infected cells by counting over 500 DAPI positive cells for each condition.

Cuevas R.A., Ghosh A., Wallerath C., Hornung V., Coyne C.B, & Sarkar S.N. (2016). MOV10 provides antiviral activity against RNA viruses by enhancing RIG-I-MAVS-independent IFN induction. Journal of immunology (Baltimore, Md. : 1950), 196(9), 3877-3886.

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Visualizing Psoralen-Induced DNA Crosslinks

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To generate localized psoralen-induced intrastrand crosslinks and ICLs, U2OS and YFP-CSB^del expressing CS1AN cells were grown on 24 mm coverslips and treated with 50 μM of 8-MOP for 2 h. A 355-nm UV-A laser, set at 24% intensity laser power and low (8%) speed, attached to a PALM laser dissection microscope (Zeiss) equipped with a 40× 0.60 NA Korr LD Plan Neofluar objective, was used to activate 8-MOP in a track along cell nuclei. Irradiated cells were fixed with 2% paraformaldehyde supplemented with 0.1% Triton X-100 and washed in PBS buffer (PBS containing 0.15% glycine and 0.5% BSA) after which immunofluorescence was performed as described (28 (link)). Antibodies used were against CSB (E-18, 1:250, Santa Cruz, sc-10459), FANCD2 (FI-17, 1:1000, Novus Biologicals, nb100-316), XPA (FL-273, 1:100, Santa Cruz, sc-853), γH2AX (JBW301, 1:1000, Millipore, 05-636). Secondary antibodies were conjugated with Alexa Fluor 488, 555 and 633 (Invitrogen). DAPI vectashield (Vector Laboratories) was used to mount coverslips. Slides were imaged using an LSM700 confocal microscope (Zeiss).

Slyskova J., Sabatella M., Ribeiro-Silva C., Stok C., Theil A.F., Vermeulen W, & Lans H. (2018). Base and nucleotide excision repair facilitate resolution of platinum drugs-induced transcription blockage. Nucleic Acids Research, 46(18), 9537-9549.

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Quantifying TUNEL-Positive Apoptotic Cells

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TUNEL assays were carried out using a DeadEnd Fluorometric TUNEL system (Promega, Madison, WI) per the manufacturer’s instructions. Cell nuclei that were fluorescently stained with FITC (green) were defined as TUNEL-positive nuclei. Slides were coverslipped with 4,6-diaminidino-2-phenylindole (DAPI) Vectashield (Vector Laboratories, Burlingame, CA). TUNEL-positive nuclei were monitored using fluorescence microscopy (Nikon, Tokyo, Japan). Five randomly selected microscopic fields were quantitatively analyzed using ImageJ.

Miao L., Li J., Liu Q., Feng R., Das M., Lin C.M., Goodwin T.J., Dorosheva O., Liu R, & Huang L. (2017). Transient and Local Expression of Chemokine and Immune Checkpoint Traps To Treat Pancreatic Cancer. ACS nano, 11(9), 8690-8706.

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Quantifying Renal Apoptosis via TUNEL

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TUNEL assays were performed to detect DNA strand breaks using a commercial kit following the instructions provided by the manufacturer's (Roche's In Situ Cell Death Detection Kit, Fluorescein (Indianapolis, IN)) recommendations. Briefly, 15-μm-thick sections of renal (n=6 per group) were mounted onto Silane-coated glass slides. Slides were deparaffinized, rehydrated, put into 10mM citrate pH 6 in a 95° water bath for 30 minutes for permeabilization and further digested with 1μg/ml proteinase K for 10 minutes at 37°. TUNEL reagents were applied to the slides according to the manufacturer's instructions. Then they were mounted with DAPI Vectashield (Vector Laboratories). Controls for this procedure included a slide where the TdT enzyme was omitted and another where the slide was pretreated with DNAse I before the normal TUNEL procedure. Photographs of sections were captured using CCD camera (Leica DC300F). The number of apoptotic nuclei was counted in four different fields and mean was found by using the image analysis software 'Leica Qwin'. Percentage of TUNEL positive cells was calculated on the number of TUNEL positive cells out of 100 total cells that were counted. Student's t-test was used to determine statistical significance levels (P≤0.05).

Yang M., Zhuang Y.Y., Wang W.W., Zhu H.P., Zhang Y.J., Zheng S.L., Yang Y.R., Chen B.C., Xia P, & Zhang Y. (2018). Role of Sirolimus in renal tubular apoptosis in response to unilateral ureteral obstruction. International Journal of Medical Sciences, 15(13), 1433-1442.

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Pancreatic Tissue Immunohistochemistry Protocol

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Pancreatic tissue samples were fixed overnight in 4% formaldehyde (Klinipath), stored in 70% ethanol, and subsequently embedded in paraffin. After deparaffinization and rehydration in xylene and ethanol, respectively, antigen retrieval was performed in citric buffer for 20 min. Sections were blocked with 2% normal donkey serum and 1% lamb serum in PBS. Primary antibodies were rabbit anti-Ftl (ab69090), mouse anti-glucagon (ab10988), and guinea pig anti-insulin (ab7842). Alexa Fluor-conjugated secondary antibodies against rabbit, mouse, and guinea pig immunoglobulin G (IgG) (Life Technologies; A11008, A10037, and A21450) were used at a dilution of 1:200. Nuclear counterstaining was done by embedding with DAPI Vectashield (Vector Laboratories, H-1500). Imaging was performed on a Leica SP8 confocal microscope using hybrid detectors.

Grün D., Muraro M.J., Boisset J.C., Wiebrands K., Lyubimova A., Dharmadhikari G., van den Born M., van Es J., Jansen E., Clevers H., de Koning E.J, & van Oudenaarden A. (2016). De Novo Prediction of Stem Cell Identity using Single-Cell Transcriptome Data. Cell Stem Cell, 19(2), 266-277.

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