Vectashield antifade mounting medium

Manufactured by Vector Laboratories

Sourced in United States, United Kingdom, Germany, Canada

Vectashield antifade mounting medium is a water-based solution designed to reduce photobleaching of fluorescent samples during microscopic examination. It is formulated to maintain the brightness and clarity of fluorescent signals.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (42 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (29 mentions) Lsm 710 confocal microscope, by Zeiss (24 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (21 mentions) Hoechst 33342, by Thermo Fisher Scientific (19 mentions) Lsm 880, by Zeiss (16 mentions) Alexa fluor 488, by Thermo Fisher Scientific (16 mentions) Lsm 880 confocal microscope, by Zeiss (14 mentions) Lsm 700, by Zeiss (14 mentions) Triton x 100, by Merck Group (12 mentions) Lsm 780 confocal microscope, by Zeiss (12 mentions) Bovine serum albumin (bsa), by Merck Group (8 mentions) Lsm 780, by Zeiss (7 mentions) Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (7 mentions) Airyscan, by Zeiss (6 mentions) Superfrost plus slide, by Thermo Fisher Scientific (6 mentions) Lsm 700 confocal microscope, by Zeiss (6 mentions) Axio observer z1, by Zeiss (6 mentions) Axiovert 200, by Zeiss (5 mentions) Tcs sp8 confocal microscope, by Leica (5 mentions)

Close spelling variants of this product

Vectashield antifade mounting medium with DAPI (H-1900, VECTASHIELD Antifade Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) VECTASHIELD PLUS Antifade Mounting Medium with DAPI Vectashield HardSet Antifade Mounting Medium with DAPI (H-1500) VECTASHIELD Antifade Mounting Medium with DAPI mounting buffer VECTASHIELD HardSet Antifade Mounting Medium with DAPI Vectashield antifade mounting medium for fluorescence Vectashield Vibrance Antifade Mounting Medium with 4′,6‐diamidino‐2‐phenylindole (DAPI, Vectashield antifade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) VECTASHIELD Antifade Mounting Medium with DAP

516 protocols using vectashield antifade mounting medium

Visualizing Protein Localization and Dynamics in Cells

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To detect protein localization by immunofluorescence in fixed cells, cells were seeded on High Performance no.1.5 18 × 18 mm glass coverslips and were fixed for 10 min, followed by permeabilization with 0.5% Triton X-100 for 5 min. Then cells were blocked with 1% BSA for 1 h at room temperature. Primary antibodies were diluted with 1% BSA (SC35 sigma S4045 1:400, RPA194 Santa Cruz sc-48385 1:200, coilin sigma C1862 1:400) and incubated for 1 h at room temperature. After washing with 1× DPBS three times, fluorescent secondary antibodies (Invitrogen A-21424 or A-21236) were 1:1000 diluted in 1% BSA and incubated for 1 h at room temperature. Samples were mounted in VECTASHIELD antifade mounting medium (Vector Lab).
To detect EGFP -tagged proteins in fixed cells, cells were seeded on High Performance no.1.5 18 × 18 mm glass coverslips and were fixed for 10 min at room temperature. Samples were mounted with VECTASHIELD antifade mounting medium (Vector Lab).
For live-cell imaging, cells were seeded on 35 mm no.1.5 glass bottom dishes (Cellvis) 1 d prior to imaging. Cells were washed once with PBS and the medium was replaced by FluoroBrite DMEM (Gibco) supplemented with 10% FBS and placed back in the incubator for 1 h. All images were obtained at 37°C with 5% CO₂ condition.

Yao R.W., Luan P.F, & Chen L.L. (2021). An optimized fixation method containing glyoxal and paraformaldehyde for imaging nuclear bodies. RNA, 27(6), 725-733.

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Microscopic Visualization and Quantification of DNA Damage and R-Loops

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Cells were grown onto poly-lysine (Sigma-Aldrich P7280) coated coverslips, fixed in 4% (v/v) formaldehyde and processed as previously described (Kantidakis et al., 2016 (link)). Briefly, the primary antibody, used in 1:1000 dilution was anti 53BP1 (Abcam, ab 36823) and the secondary antibody anti-rabbit Alexa Fluor 594 (Life Technologies). The coverslips were mounted using Vectashield Antifade Mounting Medium containing DAPI (Vector Laboratories, H-1200) and visualized using a Zeiss fluorescent microscope with a 63x/1.4 oil immersion and quantified with ImageJ.
For R-loop detection, cells were grown on coverslips, fixed and permeabilized in 100% ice cold methanol and acetone for 10 min and 1 min on ice, respectively, and processed as previously described (Sridhara et al., 2017 (link)). Briefly, the primary antibody, S9.6 was used in 1:500 dilution, and the secondary antibody anti-mouse Alexa Fluor 594 (Life Technologies). The coverslips were mounted using Vectashield Antifade Mounting Medium containing DAPI (Vector Laboratories, H-1200) and visualized using a Zeiss fluorescent microscope with a 63x/1.4 oil immersion and quantified with ImageJ.

Zatreanu D., Han Z., Mitter R., Tumini E., Williams H., Gregersen L., Dirac-Svejstrup A.B., Roma S., Stewart A., Aguilera A, & Svejstrup J.Q. (2019). Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability. Molecular Cell, 76(1), 57-69.e9.

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Fluorescence Resonance Energy Transfer Analysis of Protein Interactions

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The proteins studied in this work (IAPP, Aβ, and PrP) were fused to one of the fluorescent proteins (CFP or YFP). A confocal laser-scanning microscope Leica TCS SP5 (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) was used to examine colocalization and the possibility of physical interaction by the acceptor photobleaching FRET (AB FRET) method of the studied proteins. The FRET efficiency was measured as described previously by Rubel et al. [23 (link)], using Leica LAS AF X 3.7.2.22383 software (Leica Microsystems Wetzlar GmbH, Germany). CFP, or proteins fused with it in the FRET study acted as a donor (Excitation (Ex) = 458 nm; Emission (Em) = 461–510 nm). YFP or proteins fused with it in the FRET study, acted as an acceptor (Ex = 514 nm; Em = 518–580 nm). Acceptor photobleaching was performed using a 514 nm laser beam at 100% intensity.
Polylysine glass microscope slides from Gerhard Menzel GmbH (Braunschweig, Germany) were used for the FRET experiments. Preliminary yeast cells were washed three times with sterile water, then put onto a glass microscope slide, air-drying, enclosed into Antifade Mounting Medium VECTASHIELD (Vector Laboratories Inc., Newark, CA, USA), and covered with a coverslip (Gerhard Menzel GmbH, Germany).
Confocal microscope data were analyzed using “LAS AF Application Wizard Version 1.7.0” software (Leica Microsystems Wetzlar GmbH, Germany).

Kachkin D.V., Lashkul V.V., Gorsheneva N.A., Fedotov S.A., Rubel M.S., Chernoff Y.O, & Rubel A.A. (2023). The Aβ42 Peptide and IAPP Physically Interact in a Yeast-Based Assay. International Journal of Molecular Sciences, 24(18), 14122.

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Immunocytochemistry of T. vaginalis Cytoskeleton

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Trophozoites and amoeboid forms of T. vaginalis were adhered to glass slides, and then incubated with 100% methanol at -20ºC for 10 min. After 1 h incubation in a blocking buffer (phosphatebuffered saline (PBS: 1.7 mM KCl, 137 mM NaCl, 2 mM KH 2 PO 4 , and 10 mM Na 2 HPO 4 , pH 7.3), 5% goat serum, and 3% bovine serum albumin (BSA)), the cells were reacted with anti-Tvαactinin 2 polyclonal antibodies (1:800 dilution) [17] at 4°C. Following three washes with PBS, the cells were reacted with Alexa-Fluora 555-conjugated anti-rat IgG (1:400 dilution; Life Technologies, USA) at 37°C for 2 h. The slides were washed with PBS and mounted with 1.5 μg/ml 4'6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) anti-fade mounting medium (VECTASHIELD; Vector Laboratories, USA). The cells were then observed under an Axiovert 200 fluorescent microscope or LSM710 confocal microscopy (Carl Zeiss, Germany).

Lee H.Y., Kim J, & Park S.J. (2017). Role of α-Actinin 2 in Cytoadherence and Cytotoxicity of Trichomonas vaginalis. Journal of microbiology and biotechnology, 27(10).

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Quantification of γH2AX DNA Damage Foci

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We utilised a semi-automated image capture and processing software (IMARIS, UK) for γH2AX foci quantification to maintain scoring consistency in our clinical samples. Per patient sample, we spotted 5 × 10⁵ cells on each positive-charged slide (VWR International, PA). Cells were fixed using 2% paraformaldehyde/PBS for 15 min, permeabilised using 0.5% Triton-X/PBS for 15 min, and blocked with 3% bovine serum albumin/PBS for 30 min. Cell were then incubated with the following primary and secondary antibodies for 1 h for immunostaining: anti-γ-H2AX mouse monoclonal antibody (1:500 dilution in 1% BSA/PBS, Merck, MA - #2652964, #2854120), Alexa Fluor 488 secondary antibody (1:500 dilution, Molecular probe, Life Technologies, CA) in dark. Additional three washes with PBS were performed prior to mounting using Vectashield Antifade Mounting Medium mixed with 4′,6-diamidino-2-phenylindole (DAPI) (Vector laboratories, CA).
Slides were scanned with the LAS X software on a confocal laser microscope- Leica TCS SP8 (Leica microsystems, Germany). Bitplane Imaris software v8.2 was used to stack the multiplane images (minimum of 16 planes per sample), and to process the Z-stacked images for foci scoring using a uniform threshold parameter (0.5 μm cut-off for foci diameter). All irradiated and control samples were performed in duplicates, with a minimum of 50 cells scored per sample.

Chua K.L., Yeo E.L., Shihabudeen W.A., Tan S.H., Shwe T.T., Ong E.H., Lam P.Y., Soo K.C., Soong Y.L., Fong K.W., Tan T.W., Wee J.T, & Chua M.L. (2018). Intra-patient and inter-patient comparisons of DNA damage response biomarkers in Nasopharynx Cancer (NPC): analysis of NCC0901 randomised controlled trial of induction chemotherapy in locally advanced NPC. BMC Cancer, 18, 1095.

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Visualizing Cytoskeletal Alterations in Drug-Resistant Cells

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Cellular morphology and alterations in actin cytoskeleton in neratinib sensitive and resistant cells or in cells with altered integrin β3 expression were visualised by phalloidin staining as described previously [44 (link)], with minor modifications. Briefly, 1 × 10⁵ cells/500 µL well were seeded in multi-chambered glass slides (Lab-Tek II Chamber Slide, Thermo Fischer Scientific, #154534) in DMEM supplemented with 10% FBS and 1% P/S and grown at 37 °C until subconfluent. For weakly adherent human BT474 and BT474NR cells, the chamber slides were pre-coated with 100 µL/well of poly-L-lysine (Sigma, #P4707) to aid attachment. Subsequently, the cells were fixed with pre-warmed 4% (w/v) paraformaldehyde (PFA) for 10 min at RT and gently washed 3 times with PBS. Fixed cells were permeabilised with 0.1% Triton X-100 for 5 min at RT and stained with 100 µL/well of Alexa-488 conjugated phalloidin solution (1/500 dilution, Abnova, #U0281) containing 0.5 µg/mL DAPI to visualise the nuclei for 90 min at RT in the dark on a rocker. Stained cells were washed gently 3 times with PBS and slides were mounted using the Vectashield Antifade Mounting Medium (Vectorlabs, #H-1000). Fluorescent and brightfield images were taken with a Zeiss Axio Observer Inverted Microscope using the pre-set eGFP, DAPI and brightfield filters.

Nagpal A., Needham K., Lane D.J., Ayton S., Redvers R.P., John M., Selistre-de-Araujo H.S., Denoyer D, & Pouliot N. (2023). Integrin αvβ3 Is a Master Regulator of Resistance to TKI-Induced Ferroptosis in HER2-Positive Breast Cancer. Cancers, 15(4), 1216.

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Immunofluorescent Staining of Frozen Tissue Sections

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Organs were snapped frozen and 6 microns sections used for immunofluorescent staining as previously described (10 (link)). Sections were flat-mounted with VECTASHIELD antifade mounting medium (Vector Labs). Antibody mixes are shown in Supplementary Table 1. Images were acquired using a Zeiss AxioScan.Z1 microscope and analysed using ZEN software.

Bourne J.H., Beristain-Covarrubias N., Zuidscherwoude M., Campos J., Di Y., Garlick E., Colicchia M., Terry L.V., Thomas S.G., Brill A., Bayry J., Watson S.P, & Rayes J. (2021). CLEC-2 Prevents Accumulation and Retention of Inflammatory Macrophages During Murine Peritonitis. Frontiers in Immunology, 12, 693974.

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Immunofluorescence Analysis of Submandibular Gland Tissue

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Paraffin embedded submandibular gland tissue sections were processed for immunofluorescence analysis as previously described [22 (link)]. Primary antibodies used at the indicated dilutions include ΔNp63 (1:50, Cell Signaling Technology, D2K8X), K5 (1:100, gift from Dr. Julie Segre), K14 (1:100 [45 ]), alpha-smooth muscle actin (Sma) (1:200, Sigma, 1A4), Aqp5 (1:100, Alomone Labs), K7 (1:50, Abcam), Mist1 (1:100, Abcam), Troma-III (K19, 1:50, Development Studies Hybridoma Bank), Ki67 (1:100, Leica Biosystems, MM1), and Cleaved Caspase-3 (1:100, Cell Signaling Technology, D175). Sections were stained with TOPRO (Invitrogen) and mounted using VECTASHIELD Antifade Mounting Medium (Vector Laboratories) and imaged using an Andor Dragonfly Spinning Disk Confocal Microscope with Fiji [46 (link)]. Microscopy data in this study was acquired at the Optical Imaging and Analysis Facility, School of Dental Medicine, State University of New York at Buffalo.

Wrynn T., Min S., Horeth E., Osinski J., Sinha S, & Romano R.A. (2024). ΔNp63 regulates Sfrp1 expression to direct salivary gland branching morphogenesis. PLOS ONE, 19(5), e0301082.

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Drosophila Brain Immunohistochemistry

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Adult fly heads were fixed with 4% p-formaldehyde (pH 7.5) for 30–40 min at room temperature. Brains were dissected and rinsed four times in PT buffer (PBS with 0.1% Triton X-100) for 30 min. Samples were blocked in 7% normal goat serum (in PT) for 1 h, and incubated with primary antibodies at room temperature for 2 days. The primary antibodies employed were chicken anti-GFP 1:500 (Aves Labs, Inc, USA), rabbit anti-DsRed 1:500 (Clontech, USA) and homemade rat anti-Drosophila-PDF 1:500 (Depetris-Chauvin et al., 2011 (link)). Samples were washed 4 x 15 min in PT, and incubated with secondary antibody at 1:250 for 2 h at room temperature. Secondary antibodies were washed 4 x 15 min in PT and mounted in Vectashield antifade mounting medium (Vector Laboratories, USA). The secondary antibodies used were Cy2-conjugated donkey anti-rabbit, Alexa Fluor 647-conjugated AffiniPure donkey anti-rat and Cy3-conjugated AffiniPure donkey anti-rabbit (Jackson ImmunoResearch, USA). Images were taken on a Zeiss LSM 710 confocal microscope.

Herrero A., Duhart J.M, & Ceriani M.F. (2017). Neuronal and Glial Clocks Underlying Structural Remodeling of Pacemaker Neurons in Drosophila. Frontiers in Physiology, 8, 918.

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Zebrafish Retina Dissection and Fixation

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Adult Zebrafish retinas were collected, anesthetized using MESAB (0.2% ethyl‐m‐aminobenzoate methanesulfonate) (Sigma‐Aldrich), and killed by cutting their heads. Retinas were dissected using forceps and fixed in 4% paraformaldehyde (PFA) in phosphate‐buffered saline (PBS) for 2 hr at room temperature. Retinas were then cut, placed on a drop of Vectashield antifade mounting medium (Vector Labs) on top of a glass slide, and flattened using a coverslip.

Crespo C., Soroldoni D, & Knust E. (2018). A novel transgenic zebrafish line for red opsin expression in outer segments of photoreceptor cells. Developmental Dynamics, 247(7), 951-959.

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