Slowfade gold antifade

Manufactured by Thermo Fisher Scientific

Sourced in United States

SlowFade Gold Antifade is a reagent used in fluorescence microscopy to slow the photobleaching of fluorescent dyes. It helps maintain the brightness and stability of fluorescent signals during imaging.

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Lab products found in correlation

Lsm 710 confocal microscope, by Zeiss (6 mentions) Triton x 100, by Merck Group (6 mentions) Lsm 880 confocal microscope, by Zeiss (6 mentions) Bovine serum albumin (bsa), by Merck Group (5 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (5 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Hoechst 33342, by Thermo Fisher Scientific (3 mentions) Paraformaldehyde solution, by Electron Microscopy Sciences (3 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (3 mentions) Triton x 100 solution, by Merck Group (3 mentions) Anti h2b, by Merck Group (2 mentions) Alexa 568 anti mouse igg, by Thermo Fisher Scientific (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Phalloidin alexa fluor 488 647, by Thermo Fisher Scientific (2 mentions) Tcs sp8 microscope, by Leica (2 mentions) Tritc conjugated phalloidin, by Merck Group (2 mentions) Alexa fluorophore, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Sytox green, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 conjugate, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

47 protocols using slowfade gold antifade

Immunofluorescence Staining of Aneuploid Cells

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HCT116 and RPE1 cells and their aneuploid derivatives were seeded to a 96-well plate (HCT116: 10⁴ cells per well, RPE1: 10³ cells per well) and incubated at 37 °C and 5% CO₂ on the day before experiments. For cell fixation, a 3% formaldehyde solution was used for 15 min at room temperature. Next, cells were permeabilized with 0.1% Triton for 20 min at room temperature. Then, cells were preblocked with 3% bovine serum for 30 min at room temperature. Primary antibodies were diluted in blocking solution (Supplementary table 2) and incubated overnight at 4 °C. The next day, cells were washed and secondary antibodies were added in a concentration of 1 mg per ml followed by incubation for 1 h in the dark at room temperature. SYTOX® green (Invitrogen) or DAPI (Invitrogen) were used to localize the nucleus. For cytoplasmic staining, the HCS Cell Mask (H327 12 Component, 701618, Invitrogen) was applied. The cells were covered with SlowFade Gold Antifade (Invitrogen). To localize mitochondria, MitoTracker^TM Red CMXRos (M7512, Invitrogen) was used (100 nM for 45-h incubation at 37 °C). For lysosome localization, we also used LysoTracker^TM Red DND-99 (L7528, Thermofisher Scientific) according to the manufacturer’s protocol. Information regarding the used antibodies can be found in Supplementary table 2.

Krivega M., Stiefel C.M., Karbassi S., Andersen L.L., Chunduri N.K., Donnelly N., Pichlmair A, & Storchová Z. (2021). Genotoxic stress in constitutive trisomies induces autophagy and the innate immune response via the cGAS-STING pathway. Communications Biology, 4, 831.

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Xenograft Zebrafish Model for Cancer

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In vivo zebrafish experiments were performed in the zebrafish core facility at the University of Helsinki. All procedures were approved by the ethical committee of the regional state administrative agency (license ESAVI/13139/04.10.05/2017). Two-day post-fertilization zebrafish larvae were dechorionated and anaesthetized using 0.04% Tricaine (n = 10 per matrix group). Fluorescently labelled with CellTrace^™ Far Red (Thermo Fisher Scientific; Waltham, MA, USA), HSC-3 cells were washed in PBS and resuspended in 1:1 Matrigel or Myogel, and then microinjected into the perivitelline space using glass microinjection needles (about 1000 cells). Fish were maintained at 34 °C within an embryonic medium (Sigma-Aldrich, St. Louis, MO, USA) for 72 h and then collected, fixed with 10% PFA, and mounted using SlowFade Gold Antifade (Invitrogen, Carlsbad, CA, USA).

Hujanen R., Almahmoudi R., Salo T, & Salem A. (2021). Comparative Analysis of Vascular Mimicry in Head and Neck Squamous Cell Carcinoma: In Vitro and In Vivo Approaches. Cancers, 13(19), 4747.

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Immunofluorescence Staining Protocol

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Slides were prepared as for DNA FISH. Cells were permeabilized in 0.1% Triton-PBS for 15 minutes, then washed three times in PBS-T (PBS with 0.1% Tween 20) for 10 minutes each. Proteins were blocked in 1% bovine serum albumin (BSA) in PBS-T for 1 hour at RT. Primary antibodies diluted in 1% BSA-PBS-T were added to the slide, covered with coverslip, and sealed with rubber cement. Slides were transferred to humidified chamber and incubated overnight at 4°C. The following day, slides were washed three times in PBS-T for 10 minutes each. Secondary antibody was diluted in 1% BSA-PBS-T, added to slide, covered with coverslip, and sealed with rubber cement. Slides were transferred to humidified chamber and incubated at RT for 2 hours. Slides were then washed twice in PBS-T for 10 minutes each, and once in PBS for 10 minutes. To stain DNA, slides were washed with Hoechst (1:10,000 in 2× SSC) for 5 minutes. Slides were then mounted in SlowFade Gold Antifade (Invitrogen).

Luppino J.M., Park D.S., Nguyen S.C., Lan Y., Xu Z., Yunker R, & Joyce E.F. (2020). Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes. Nature genetics, 52(8), 840-848.

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Senescence Detection in Wing Discs

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CellEvent senescence detection kit from Invitrogen (C10850) was used to check SA-β-gal activity, following the instructions of manufacturer. Wing discs were dissected in PBS, fixed with 4% PFA, washed with 1% BSA (in PBS), and then incubated in working solution for 2 h at 37°C. Washing steps were performed in PBS and PBS containing 0.1% TritonX-100 (PBT). Tissues were counterstained with DAPI (0.25 ng/μl, 520 Sigma, D9542). Tissues were mounted using SlowFade Gold Antifade (Invitrogen, S36936).

Jaiswal J., Egert J., Engesser R., Peyrotón A.A., Nogay L., Weichselberger V., Crucianelli C., Grass I., Kreutz C., Timmer J, & Classen A.K. (2023). Mutual repression between JNK/AP-1 and JAK/STAT stratifies senescent and proliferative cell behaviors during tissue regeneration. PLOS Biology, 21(5), e3001665.

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Nucleolin and H2B Immunostaining Protocol

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Cells grown on coverslips (18 × 18 or 22 × 22 mm²) were briefly washed twice using 1X Phosphate Buffered Saline (PBS) and fixed for 10 min in 4% Paraformaldehyde (Sigma, P6148) in PBS, pH 7.4 at RT, washed thrice in 1X PBS (5 min each), followed by permeabilization in 0.5% Triton-X-100 (in PBS) for 10 min at RT. Cells were blocked in 1% Bovine serum albumin (BSA) (Sigma, A2153) in 1X PBS, for 30 min, washed thrice with 1X PBS and incubated in primary antibodies for 90 min at RT and secondary antibodies for 60 min at RT, with washes in between using 1X PBS. Primary antibody was diluted in 0.5% BSA (prepared in 1X PBS): anti-Nucleolin (ab13541), 1:300, anti-H2B (Millipore, 07-371), 1:100. Following secondary antibodies were used after diluting in 1X PBS +0.1%Triton X-100 (PBST): anti-mouse IgG-Alexa 568 (Molecular Probes), 1:1000; anti-rabbit IgG Alexa 488 (Molecular Probes) after which cells were washed thrice in 1X PBST. Cells were mounted in Slowfade Gold Antifade (Invitrogen, S36937). Cells were imaged on a Zeiss LSM710 confocal microscope with 405 nm, 458 nm and 561 nm laser lines at 2% power using a 63X oil immersion objective, NA 1.4 at 2.5X digital zoom. X-Y resolution: 512 pixels X 512 pixels (1 pixel = 0.11 µm). Confocal z-stacks were collected at an interval of 0.32 µm.

Sen Gupta A., Joshi G., Pawar S, & Sengupta K. (2018). Nucleolin modulates compartmentalization and dynamics of histone 2B-ECFP in the nucleolus. Nucleus, 9(1), 350-367.

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Synthesis and Characterization of Fluorescent Nanoparticles

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PMAn,
CDDP, anhydrous DMF,
silver nitrate, o-phenylenediamine, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDC), N,N-dimethyl
amino pyridine (DMAP), pyrene, ethylenediamine, rhodamine B isothiocyanate,
dimethyl sulfoxide (DMSO-d₆), methanol-d₄, and silicon wafer for FESEM were bought from
Sigma-Aldrich. PTX was purchased from Selleck Chemical. Dialysis membranes
(3.5 kDa) were purchased from Spectrum Labs. MCF7 cells were procured
from ECACC. Dulbecco’s modified Eagle’s medium (DMEM),
fetal bovine serum (FBS), LysoTracker Green DND-26, Hoechst 33342,
SlowFade Gold antifade, and Alexa Fluor-conjugated phalloidin 568
were purchased from Invitrogen. MTT reagent and tissue culture grade
DMSO were purchased from Sigma-Aldrich. 96-well flat-bottomed tissue-culture
plates were obtained from Corning.

Palvai S., Anandi L., Sarkar S., Augustus M., Roy S., Lahiri M, & Basu S. (2017). Drug-Triggered Self-Assembly of Linear Polymer into Nanoparticles for Simultaneous Delivery of Hydrophobic and Hydrophilic Drugs in Breast Cancer Cells. ACS Omega, 2(12), 8730-8740.

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SGPP1 Immunofluorescence in Myeloma Cells

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Air dried cytospins containing MM cells were fixed in 10% paraformaldehyde for 10 min followed by a treatment with cold methanol at −20 °C for 30 min and three PBS washes. Cytospins were then blocked with 5% BSA containing 0.2% Triton-100 for 1 h at room temperature. Slides were incubated with rabbit anti-human SGPP1 antibody (Sigma-Aldrich, Saint Louise, MO, USA) in PBST containing 2% BSA overnight at 4 °C in a humid container. The PBS washed slides were then incubated with Alexa Fluor 568 donkey anti-rabbit IgG (H + L), Invitrogen, Eugene, OR, USA) for 1 h. Slowfade Gold antifade (Invitrogen, Eugene, OR) containing DAPI (4, 6-diamidino-2-phenylindole) was used to mount the slides. The images were taken on an Olympus IX73 (Waltham, MA, USA) microscope.

Petrusca D.N., Mulcrone P.L., Macar D.A., Bishop R.T., Berdyshev E., Suvannasankha A., Anderson J.L., Sun Q., Auron P.E., Galson D.L, & Roodman G.D. (2022). GFI1-Dependent Repression of SGPP1 Increases Multiple Myeloma Cell Survival. Cancers, 14(3), 772.

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Immunohistochemistry of Drosophila Wing Discs

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Wing discs from third-instar larvae were dissected and fixed for 15 min at room temperature in 4% paraformaldehyde in PBS. Washing steps were performed in PBS containing 0.1% TritonX-100 (PBT). Discs were then incubated with primary antibodies in PBT, gently mixing overnight at 4°C. Tissues were counterstained with DAPI (0.25 ng/µl, Sigma, D9542), Phalloidin-Alexa Fluor 488/647 (1:100, Life Technologies) or Phalloidin-conjugated TRITC (1:400, Sigma) during incubation with cross-absorbed secondary antibodies coupled to Alexa Fluorophores (Invitrogen or Abcam) at room temperature for 2 hr. The gstD-GFP is a GFP reporter under the control of 2.7 kB upstream regulatory region of GstD as published by Dirk Bohmann’s lab (Sykiotis and Bohmann, 2008 (link)) and the Trbl GFP is a GFP-trap MIMIC line inserted in the intron of Trbl (Bloomington stock #61654). Tissues were mounted using SlowFade Gold Antifade (Invitrogen, S36936). Whenever possible, experimental and control discs were processed in the same vial and mounted on the same slides to ensure comparability in staining between different genotypes. Images were acquired using the Leica TCS SP8 Microscope, using the same confocal settings and processed using tools in Fiji. Per-channel views are shown in Figure 3—figure supplement 5.

Floc'hlay S., Balaji R., Stanković D., Christiaens V.M., Bravo González-Blas C., De Winter S., Hulselmans G.J., De Waegeneer M., Quan X., Koldere D., Atkins M., Halder G., Uhlirova M., Classen A.K, & Aerts S. (2023). Shared enhancer gene regulatory networks between wound and oncogenic programs. eLife, 12, e81173.

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Immunofluorescent Staining of Mitochondria

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Cells grown on glass coverslips were fixed for 1 h at room temperature (RT) using 4% paraformaldehyde (PFA) dissolved in phosphate buffered saline (PBS), washed, and then incubated for 30 min at RT in a PBS-based blocking buffer containing 0.3% Triton X-100, 5% bovine serum albumin. All antibodies used were diluted in this blocking buffer. Samples were subsequently incubated overnight at 4 °C with TOMM20 (1:200) rabbit polyclonal antibody (Catalog number - FL-145; C11415; Santa Cruz Biotechnology; Santa Cruz, CA), washed, and incubated with DyLight 488- conjugated AffiniPure Goat Anti-Rabbit IgG (1:400) (RRID: AB_2339507; Jackson ImmunoResearch Laboratories; West Grove, PA) for 1 h at RT. Coverslips were then stained with Texas Red-X phalloidin (1:70 in PBS) (Invitrogen Molecular Probes; Eugene, OR) for 30 min at RT, washed and then stained with Dapi (1:10,000 in PBS) for 5 min. Coverslips were mounted on slides using SlowFade Gold antifade (Invitrogen; Life Technology; Carlsbad, CA), fluorescence signals visualized by confocal microscopy (Fluoview FV1000 confocal microscope, Olympus; Center Valley, PA) and images collected using the Fluoview 1000 software.

Panvini A.R., Gvritishvili A., Galvan H., Nashine S., Atilano S.R., Kenney M.C, & Tombran-Tink J. (2022). Differential mitochondrial and cellular responses between H vs. J mtDNA haplogroup-containing human RPE transmitochondrial cybrid cells. Experimental eye research, 219, 109013.

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Immunofluorescence Imaging of Nucleolin and H2B

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Cells grown on coverslips (18 × 18 or 22 × 22 mm²) were briefly washed twice using 1X Phosphate Buffered Saline (PBS) and fixed for 10 min in 4% Paraformaldehyde (Sigma, P6148) in PBS, pH 7.4 at RT, washed thrice in 1X PBS (5 min each), followed by permeabilization in 0.5% Triton-X-100 (in PBS) for 10 min at RT. Cells were blocked in 1% Bovine serum albumin (BSA) (Sigma, A2153) in 1X PBS, for 30 min, washed thrice with 1X PBS and incubated in primary antibodies for 90 min at RT and secondary antibodies for 60 min at RT, with washes in between using 1X PBS. Primary antibody was diluted in 0.5% BSA (prepared in 1X PBS): anti-Nucleolin (ab13541), 1:300, anti-H2B (Millipore, 07-371), 1:100. Following secondary antibodies were used after diluting in 1X PBS +0.1%Triton X-100 (PBST): anti-mouse IgG-Alexa 568 (Molecular Probes), 1:1000; anti-rabbit IgG Alexa 488 (Molecular Probes) after which cells were washed thrice in 1X PBST. Cells were mounted in Slowfade Gold Antifade (Invitrogen, S36937). Cells were imaged on a Zeiss LSM710 confocal microscope with 405 nm, 458 nm and 561 nm laser lines at 2% power using a 63X oil immersion objective, NA 1.4 at 2.5X digital zoom. X-Y resolution: 512 pixels X 512 pixels (1 pixel = 0.11 μm). Confocal z-stacks were collected at an interval of 0.32 μm.

Sen Gupta A., Joshi G., Pawar S, & Sengupta K. (2018). Nucleolin modulates compartmentalization and dynamics of histone 2B-ECFP in the nucleolus. Nucleus, 9(1), 350-367.

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