Antifade mounting medium with dapi

Manufactured by Vector Laboratories

Sourced in United States, United Kingdom

Antifade Mounting Medium with DAPI is a solution designed to preserve and protect fluorescent signals in microscopy samples. It contains an antifade agent to reduce photobleaching and DAPI, a fluorescent DNA-binding dye that stains nuclei.

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Lab products found in correlation

Lsm 710, by Zeiss (6 mentions) Lsm 700, by Zeiss (4 mentions) Ab7475, by Abcam (3 mentions) Cryostat, by Leica (3 mentions) Cryojane, by Leica (3 mentions) Fv1000 confocal laser scanning microscope, by Olympus (3 mentions) Alexa fluor 488, by Thermo Fisher Scientific (3 mentions) Lsm 710 confocal microscope, by Zeiss (3 mentions) Fluorescence microscope, by Zeiss (2 mentions) Cm1850 uv, by Leica (2 mentions) Tissue plus optimal cutting temperature compound, by Thermo Fisher Scientific (2 mentions) Anti mouse cd3 clone 17a2 efluor 660 antibody, by Thermo Fisher Scientific (2 mentions) Ab8898, by Abcam (2 mentions) Ab8580, by Abcam (2 mentions) Sc 6215, by Santa Cruz Biotechnology (2 mentions) Ab16048, by Abcam (2 mentions) Tissue tek oct compound, by Sakura Finetek (2 mentions) Alexa fluor 647 goat anti rabbit, by Thermo Fisher Scientific (2 mentions) Ab22552, by Abcam (2 mentions) Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)

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86 protocols using antifade mounting medium with dapi

Immunofluorescence Staining Protocol for Cultured Cells

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Cells were seeded on cover glass in a 24‐well plate and cultured for 24–48 h. After washing with PBS, cells were fixed with 4% formaldehyde in PBS for 10 min and permeabilized with 0.5% Triton‐X100 in PBS for 5 min. Cells were then washed 3 times with 0.1% PBS‐Tween (PBST), blocked with 3% BSA in PBST for 1 h, washed 3 times with 0.1% PBST and incubated with indicated primary antibody overnight at 4 °C (Table S2, Supporting Information). Cells were then washed 3 times with 0.1% PBST and incubated with indicated secondary antibodies (ThermoFisher, USA) in 3% BSA in the dark for 1 h at room temperature. After washing 3 times with 0.1% PBST, cells were mounted with Antifade Mounting Medium with DAPI (Vector laboratories) and sealed with clear nail polish. Images were captured with a fluorescence microscope (ZEISS, Germany).

Liu W., Yu X., Yuan Y., Feng Y., Wu C., Huang C., Xie P., Li S., Li X., Wang Z., Qi L., Chen Y., Shi L., Li M.J., Huang Z., Tang B., Chang A, & Hao J. (2022). CD73, a Promising Therapeutic Target of Diclofenac, Promotes Metastasis of Pancreatic Cancer through a Nucleotidase Independent Mechanism. Advanced Science, 10(6), 2206335.

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Immunofluorescence Analysis of FXR and β-Catenin

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For immunofluorescence analysis, cells were fixed with 4% paraformaldehyde in PBS at 4°C for 30 minutes, washed 3 times with PBS, and permeabilized by incubation in PBS with 0.1% Triton X-100 for 10 minutes. After blocking in PBS with 10% goat serum and 0.1% Triton X-100 for 1 hour at room temperature, cells were incubated with the primary antibodies (anti-FXR, Abmart, China) monoclonal mouse antibody and anti-β-Catenin rabbit antibody (Cell Signaling) at 4°C overnight, followed by incubation with the secondary antibodies (Alexa488-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA) and Alexa 594-conjugated anti-mouse IgG (Invitrogen, Carlsbad, CA) at room temperature for 2 hours. Coverslips were mounted onto glass slides with anti-fade mounting medium with DAPI (Vector Laboratories) and visualized with an Olympus FluoView 500 IX71 confocal microscope.

Liu X., Zhang X., Ji L., Gu J., Zhou M, & Chen S. (2015). Farnesoid X receptor associates with β-catenin and inhibits its activity in hepatocellular carcinoma. Oncotarget, 6(6), 4226-4238.

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Mouse Tail-Vein Injection for Metastatic Cancer

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All animal procedures were approved by Boston University IACUC (PROTO201800533). The mouse tail-vein injection model was used to study the development of metastatic cancer. 6-week-old male homozygous nude (Foxn1nu/Foxn1nu) mice obtained from the Jackson Laboratory were used. 1205Lu melanoma cells stabling expressing GFP (Applied Biological Materials) were transfected with lentivirus carrying negative control shRNA (1205Lu-shNC) or FADS2 targeting shRNA (1205Lu-shFADS2 #2). The cells were then prepared in sterile PBS in a completely monocellular suspension without clumps at

1 \times 10^{6} m L^{- 1}

concentration. 100 μL of cell suspension was injected slowly via the lateral tail vein of the anesthetized mouse, after which the bleeding was stopped by applying pressure to the puncture site with a dry piece of gauze. The lung tissues were collected 46 days after tumor inoculation and sliced using a cryostat at 10 to 20 μm thickness. Then, the tissues were mounted with the antifade mounting medium with DAPI (Vector Laboratories). The tissues were imaged using an Olympus VS120 Automated Slide Scanning Microscope.

Lee H.J., Chen Z., Collard M., Chen F., Chen J.G., Wu M., Alani R.M, & Cheng J.X. (2021). Multimodal Metabolic Imaging Reveals Pigment Reduction and Lipid Accumulation in Metastatic Melanoma. BME Frontiers, 2021, 9860123.

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Immunohistochemical Analysis of Glial and Neuronal Markers in Brain Tissue

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The embedded brains in OCT were cut into coronal sections (10 μm) using a Tissue-Tek cryostat and mounted onto charged glass slides. Prior to staining, slides were washed with PBS (3x, 5,5, and 10 min); then, they were treated with blocking buffer (5% normal goat serum, 0.2% Triton X-100, 0.5% bovine albumin in PBS) for 1h at room temperature. The treated sections were then incubated overnight at 4°C with primary anti-GFAP antibody (1:100 dilution, Biolegend San Diego, CA, USA) or anti-IBA1 antibody (1:250 dilution, FUJIFILM Wako Chemicals U.S.A Corporation Richmond, VA, USA). For OCTN1 staining, the slides were treated with OCTN1/2 (H-9) antibodies (1:100 dilution, Santa Cruz Biotechnology, Dallas, TX, USA). Then, the slides were washed with PBS (3x) for 10 min each, the sections stained for GFAP and OCTN1 were subsequently incubated with secondary antibody goat anti-mouse Alexa Fluor 488 (1:200 dilution, Thermo Fisher Scientific, Carlsbad, CA, USA). Sections stained for IBA1 were incubated with secondary antibody goat anti-rabbit Alexa Fluor 680 (1:200 dilution, Thermo Fisher Scientific, Carlsbad, CA, USA) for 30 min at room temperature. The sections were then washed with PBS twice for 10 minutes and once for 30 minutes, and cover slipped with an antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA) before observation under a fluorescence microscope.

Behof W.J., Whitmore C.A., Haynes J.R., Rosenberg A.J., Tantawy M.N., Peterson T.E., Harrison F.E., Beelman R.B., Wijesinghe P., Matsubara J.A, & Pham W. (2022). Improved synthesis of an ergothioneine PET radioligand for imaging oxidative stress in Alzheimer’s disease. FEBS letters, 596(10), 1279-1289.

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Detecting MERS-CoV S Protein Expression

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Indirect immunofluorescence assay (IFA) was used to detect S protein expression in rSRV9-MERS_S1-infected cells. NA cells were plated on coverslips in 35-mm-diameter dishes and infected with rSRV9-MERS_S1 or rSRV9 at a MOI = 1. At 24 h post-infection, cells were fixed in 4% paraformaldehyde (Solarbio Corporation, Beijing, China) at 4°C for 30 min and blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) at RT for 1 h. Cells were incubated with rabbit anti-MERS-CoV S protein polyclonal antibody (Sino Biologicals, Beijing, China) and mouse anti-RABV G protein monoclonal antibody (Merck Millipore, Hong Kong, China) 1:100 diluted in PBS containing 1% BSA at 37°C for 1 h. Then the cells were washed 3 times with PBST and stained with 594-conjugated goat anti-rabbit antibody (Abcam, Shanghai, China, 1:500 diluted in PBS containing 1% BSA) and 488-conjugated goat anti-mouse antibody (Abcam, Shanghai, China, 1:1,000 diluted in PBS containing 1% BSA) at 37°C for 1 h. After being washed 3 times, cells were stained with an antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Cells were analyzed with an Axio Scope.A1 microscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany), and representative images were obtained from the ZEN 2012 system (Carl Zeiss MicroImaging GmbH, Göttingen, Germany).

Chi H., Wang Y., Li E., Wang X., Wang H., Jin H., Han Q., Wang Z., Wang X., Zhu A., Sun J., Zhuang Z., Zhang L., Ye J., Wang H., Feng N., Hu M., Gao Y., Zhao J., Zhao Y., Yang S, & Xia X. (2022). Inactivated Rabies Virus Vectored MERS-Coronavirus Vaccine Induces Protective Immunity in Mice, Camels, and Alpacas. Frontiers in Immunology, 13, 823949.

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Immunohistochemical Analysis of Neuroinflammation

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Brains from 5xFAD mice and WT mice were fixed in 4% paraformaldehyde overnight at 4°C followed by 30% sucrose solution for 2 days at 4°C. Coronal slices with a thickness of 35 µm were obtained by using a cryostat (Leica CM1850, Wetzlar, Germany) and were permeabilized with PBS containing 0.2% Triton X-100 (PBST) and 10% normal goat serum for 1 h at room temperature. The sections were then immunostained with an anti-NRF2, anti-Iba-1, anti-GFAP, anti-NLRP3, anti-cleaved caspase-1, anti-IL-1β, or anti-IL-6 antibody diluted in PBST with 10% normal goat serum for 24–48 h at 4°C. The sections were washed three times in PBST, incubated with the corresponding secondary antibody at room temperature for 2 h, washed three times with PBS, and mounted in antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Fluorescence microscopy was used to acquire images of the sections (DMi8, Leica Microsystems, Wetzlar, Germany), which were analyzed by the software ImageJ (version 1.53a, National Institutes of Health, Bethesda, MD, USA). The primary and secondary antibodies are detailed in Table 1.

Lee H.J., Park J.H, & Hoe H.S. (2022). Idebenone Regulates Aβ and LPS-Induced Neurogliosis and Cognitive Function Through Inhibition of NLRP3 Inflammasome/IL-1β Axis Activation. Frontiers in Immunology, 13, 749336.

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Visualizing ACTB mRNA Translocation

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HEK293FT cells were plated in 24-well tissue culture plates on poly-D-lysine coverslips (Corning) and transfected with 150ng dCas13a-NF vector and 300 ng guides for imaging ACTB. For translocation experiments, cells were fixed with 4% PFA and permeabilized with 0.2% Triton X-100 after 48 hours and mounted using anti-fade mounting medium with DAPI (Vectashield). Confocal microscopy was performed using a Nikon Eclipse Ti1 with Andor Yokagawa Spinning disk Revolution WD system.
Nuclear export of dCas13a-NF-msfGFP with guides targeting ACTB mRNA was analyzed by measuring the average cytoplasmic and nuclear msfGFP fluorescence and comparing the ratio across many cells between targeting and non-targeting conditions.

Abudayyeh O.O., Gootenberg J.S., Essletzbichler P., Han S., Joung J., Belanto J.J., Verdine V., Cox D.B., Kellner M.J., Regev A., Lander E.S., Voytas D.F., Ting A.Y, & Zhang F. (2017). RNA targeting with CRISPR-Cas13a. Nature, 550(7675), 280-284.

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Chromosome Visualization Using FISH

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FISH analysis was performed, as described previously [48 (link)]. The pTa794 clone containing 5S rDNA repeats or pAtT4 clone containing telomeric-specific repeats were used as FISH probes [60 (link),90 (link)]. Probes were labeled with fluorophore-dUTP (Roche, Basel, Switzerland) by nick translation. Anti-digoxigenin (Vector Laboratories) was used as the second antibody for detection with a probe of digoxigenin (DIG). Chromosomes were counterstained using Antifade Mounting Medium with DAPI (Vector laboratories). Finally, chromosome images were taken using a DS-Qi2 Microscope Camera system installed in a Ci-S-FL microscope (Nikon).

Jing J., Zhang T., Wang Y., Cui Z, & He Y. (2019). ZmRAD51C Is Essential for Double-Strand Break Repair and Homologous Recombination in Maize Meiosis. International Journal of Molecular Sciences, 20(21), 5513.

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Immunofluorescence Characterization of Glial Cells

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To verify the identity and assess the purity of isolated glial cells, immunofluorescence microscopy was used. Briefly, cells plated on poly-D-lysine-coated coverslips were fixed in 4% paraformaldehyde at room temperature for 20 min and incubated with 2% bovine serum albumin at RT for 60 min. Then, the cells were incubated with primary antibodies against GFAP (1:200, mouse, Abcam) and IBA1 (1:100, rabbit, Abcam) overnight at 4 °C. The secondary antibodies used were DyLight 488-conjugated goat anti-rabbit IgG (1:200, EarthOx) and Alexa Fluor 594-conjugated goat anti-mouse IgG (1:200, Invitrogen) and incubated at RT for 60 min. Finally, Antifade Mounting Medium with DAPI (Vector Lab, USA) was added for nuclear staining. Images of glial cells were captured using a fluorescence microscope (Zeiss, Germany).

Liu B., Luo M., Meng D., Pan H., Shen H., Shen J., Yao M, & Xu L. (2021). Tetrahydropalmatine exerts analgesic effects by promoting apoptosis and inhibiting the activation of glial cells in rats with inflammatory pain. Molecular Pain, 17, 17448069211042117.

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Quantifying DNA Damage with γH2A.X Immunostaining

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Immunofluorescence-staining of γH2A.X was performed as described before [24 (link)]. Briefly, cells were seeded on cover glasses in 24-well plates, cultured for 24–48 h, and then treated with cisplatin alone or together with Olaparib or ZC-22 for 24 h. After washing with PBS, cells were fixed by 4% PFA, permeated with 0.5% Triton-X100 in PBS, washed thrice with 0.1% PBS-Tween (PBST), blocked with 3% BSA in PBST, and then incubated with anti-γH2A.X antibody overnight at 4 °C. Cells were then washed thrice with 0.1% PBST, incubated with Alexa Fluor 488 conjugated secondary antibody (Cell Signaling, Danvers, MA, USA) for 1 h at room temperature in dark, and mounted with Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were obtained with the Olympus FV1000 laser scanning confocal microscope. At least 150 cells in three fields were counted for each repeat.

Tian C., Wei Y., Li J., Huang Z., Wang Q., Lin Y., Lv X., Chen Y., Fan Y., Sun P., Xiang R., Chang A, & Yang S. (2022). A Novel CDK4/6 and PARP Dual Inhibitor ZC-22 Effectively Suppresses Tumor Growth and Improves the Response to Cisplatin Treatment in Breast and Ovarian Cancer. International Journal of Molecular Sciences, 23(5), 2892.

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