Prolong gold anti fade agent

Manufactured by Thermo Fisher Scientific

Sourced in United States

Prolong Gold anti-fade agent is a reagent designed to retard the fading of fluorescent signals in microscopy samples. It is intended for use with a variety of fluorophores to maintain the intensity of fluorescent signals during imaging and analysis.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions) Alexa fluor 546, by Thermo Fisher Scientific (2 mentions) Triton x 100, by Merck Group (2 mentions) Lsm800 confocal microscope, by Zeiss (2 mentions) Oct compound, by Sakura Finetek (2 mentions) Anti integrin β1 antibody, by Abcam (1 mentions) Fluorescent confocal microscope, by Zeiss (1 mentions) Human serum ab, by BD (1 mentions) Tcs sp2 se confocal microscope, by Leica camera (1 mentions) Perm wash buffer, by BD (1 mentions) Cytofix cytoperm buffer, by BD (1 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions) Hoechst, by Merck Group (1 mentions) Axio imager z1, by Leica camera (1 mentions) Metamorph software, by Molecular Devices (1 mentions) Shandon cytospin 4 cytocentrifuge, by Thermo Fisher Scientific (1 mentions) Nigericin, by Bio-Techne (1 mentions) Ruthenium red, by Bio-Techne (1 mentions) Llome, by Merck Group (1 mentions) Amiodarone hydrochloride, by Bio-Techne (1 mentions)

20 protocols using prolong gold anti fade agent

Quantifying Stem Cell Proliferation on Nanofibers

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After 15 days of culture, HNDFs grown on nanofibers were fixed in
paraformaldehyde (PFA, 4%) and Triton-X (0.05%) for 10 min. Following fixation,
samples were incubated with primary antibody (rabbit polyclonal anti-Ki67 with
4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for proliferation
study or rabbit monoclonal anti-integrin β1 antibody, Abcam) and with
secondary antibody (goat anti-rabbit IgG (H+L) secondary antibody with Alexa
fluor® 546, Invitrogen) during 1 h at room temperature for both primary
and secondary antibody incubation. Following immunostaining, samples were
mounted on glass slides by using Prolong Gold anti-fade agent (Invitrogen) and
imaged on the confocal microscopy. Cell proliferation was calculated by dividing
the number of Ki-67 positive cells by the number of DAPI-positive cells. For
statistical analysis, n=5 for PCL and n=6 for
CA and CA/SPH (field of view (FOV)=25) from 3 productions for each
condition.

Ahn S., Chantre C.O., Gannon A.R., Lind J.U., Campbell P.H., Grevesse T., O’Connor B.B, & Parker K.K. (2018). Soy Protein/Cellulose Nanofiber Scaffolds Mimicking Skin Extracellular Matrix for Enhanced Wound Healing. Advanced healthcare materials, 7(9), e1701175.

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Indirect Immunofluorescence Analysis of GRP78 in Rat and Human Sperm

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Mature (caudal) rat sperm and ejaculated normal human sperm fixed in 4% paraformaldehyde were used for Indirect Immunofluorescence studies. Post fixation, the sperm were pelleted by centrifugation at 800gfor 10min at 4°C, washed thrice in 0.1M PBS and resuspended in 0.1M PBS. 10μl of this sample was smeared on poly-L-Lysine coated slides. Sperm were either permeabilized with 1% Triton X- 100 (Sigma Aldrich, St. Louis, MO, USA) and 0.5% glacial acetic acid in 0.1M PBS for 10min at RT or left unpermeabilized. They were then washed thrice with 0.1M PBS for 5min and non-specific binding was blocked by incubating the smears in0.5% BSA at RT for 1h in a humid chamber. The sperm smears were then incubated overnight at 4°C with 1:100 diluted rat anti- GRP78 antibody raised in rabbit (Sigma Aldrich, St. Louis, MO, USA). The following day, slides were washed thrice with 0.1M PBS and then incubated with FITC conjugated Goat anti Rabbit IgG (1:100) at RT for 1h in the dark. Slides were washed as mentioned above and mounted in ProLong Gold antifade agent (Invitrogen, OR, USA) and examined ona confocal fluorescent microscope (Zeiss, Jena, Germany). Smears incubated with buffer only in place of the primary antibody served as ‘Negative control’. 4′, 6-diamidino-2-phenylindole (DAPI) (300nM) (Roche, Mannheim, Germany) was used to stain the nuclei.

Lobo V., Rao P., Gajbhiye R., Kulkarni V, & Parte P. (2015). Glucose Regulated Protein 78 Phosphorylation in Sperm Undergoes Dynamic Changes during Maturation. PLoS ONE, 10(11), e0141858.

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Immunofluorescence Analysis of CAR-T Cells

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CAR⁺ and CAR⁺ TK + ffLuc + T cells were fixed and permeabilized in 100 μl of Cytofix/Cytoperm buffer for 20 min at 4 °C (BD Biosciences) followed by washing in Perm/wash buffer (1×; BD Biosciences, CA) containing 10 % heat-inactivated human serum AB (Gemini Bio-Products, West Sacramento, CA, USA). Cells were then stained with the anti-FLAG APC mAb (PhycoLink) at a 1:200 dilution for 20 min at 4 °C, washed twice in Perm/wash buffer (1×; BD Biosciences, CA) containing 10 % heat-inactivated human serum AB. After washing, the cells were transferred onto slides in 20 μl of Prolong Gold anti-fade agent containing DAPI (Invitrogen) and cover slips were mounted and sealed along the edge. After drying at room temperature, cells were examined under a Leica TCS SP2-SE confocal microscope using oil immersion lens (×63 objective). Single-scan images were acquired in a 1024 × 1024 format with a line averaging of 8 and overlaid.

Najjar A.M., Manuri P.R., Olivares S., Flores L I.I., Mi T., Huls H., Shpall E.J., Champlin R.E., Turkman N., Paolillo V., Roszik J., Rabinovich B., Lee D.A., Alauddin M., Gelovani J, & Cooper L.J. (2016). Imaging of Sleeping Beauty-Modified CD19-Specific T Cells Expressing HSV1-Thymidine Kinase by Positron Emission Tomography. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging, 18(6), 838-848.

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Immunofluorescence Staining of RhoB

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Anti-RhoB and/or anti-Flag primary antibodies and Alexa Fluor secondary antibodies (Life Technologies) were used. Cells were fixed in 3.2% paraformaldehyde and permeabilized with 0.2% Triton X-100. Nuclei were labeled with Hoechst (Sigma-Aldrich). After mounting with ProLong Gold anti-fade agent (Invitrogen, Molecular Probes), cells were visualized using a Zeiss Axioimager Z1 or a Leica SP5 confocal microscope coupled to the MetaMorph software (Molecular Devices).

Arsic N., Ho-Pun-Cheung A., Evelyne C., Assenat E., Jarlier M., Anguille C., Colard M., Pezet M., Roux P, & Gadea G. (2017). The p53 isoform delta133p53ß regulates cancer cell apoptosis in a RhoB-dependent manner. PLoS ONE, 12(2), e0172125.

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Immunofluorescence Microscopy of Fixed Cells

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Once collected, the cells were centrifuged (on Thermo Scientific Shandon Cytospin 4 Cytocentrifuge, 5 min at 1000 rpm) and fixed [4% paraformaldehyde (PFA), 10 min]. After permeabilization (0.5% NP-40 for 5 min), the cells were washed [phosphate-buffered saline (PBS)–0.01% Triton], blocked (3% bovine serum albumin, 1 hour), and subsequently incubated with primary antibodies (see Key Resources Table) in blocking buffer. After washing (PBS–0.01% Triton), the corresponding secondary antibodies were added. Glass coverslips were then added on cells already mounted with either Mowiol (Calbiochem) or ProLong Gold Antifade agent (Invitrogen) containing DAPI. Images were taken with a ×63 objective using Zeiss epifluorescence microscope and/or confocal microscopy (Nikon spinning disk). Image analysis was performed using ImageJ software.

Compe E., Pangou E., Le May N., Elly C., Braun C., Hwang J.H., Coin F., Sumara I., Choi K.W, & Egly J.M. (2022). Phosphorylation of XPD drives its mitotic role independently of its DNA repair and transcription functions. Science Advances, 8(33), eabp9457.

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Inflammasome Activation Assay Protocol

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Uric acid, Cytochalasin D, Cathepsin B, LLOMe, KCl and ultrapure LPS were from Sigma. Nigericin, Ruthenium Red and Amiodarone Hydrochloride were purchased from Tocris. Monosodium urate crystals were made as described in Ng et al49 (link). Silica particles were purchased from US Silica Company. Crystals were added to tissue culture grade endotoxin-free water to get the stock solutions for experiments. Mouse Anti-ASC (SC-22514-R), Mouse Anti-Casp-1 (SC-514), Mouse Anti-Cath B (SC-6493) antibodies were purchased from Santa Cruz. Mouse Anti-Casp-1 P20 subunit (AG-20B-0042) was purchased from Adipogen. Anti Casp-1 antibody for Western blotting was from Abcam (EPR4321). HRP-conjugated secondary antibodies and Anti-Rabbit Dylight 549 were purchased from Jackson ImmunoResearch and eBiosciences. Alexa-conjugated secondary antibodies and ProLong Gold antifade agent were purchased from Invitrogen. Mouse Pan-CD45 (17-0451) and LY6G (11-5931) antibodies were from eBioscience. GAPDH antibody (CST 2118) was from Cell Signaling. Mouse Anti IL-1β antibody (AF-401-NA) was from R&D systems. IL-1β and TNFα ELISA ready-set-go kits were from eBioscience Inc. L cell-conditioned media for BMMAC culture was a gift from Robin Yates of the University of Calgary.

Hari A., Zhang Y., Tu Z., Detampel P., Stenner M., Ganguly A, & Shi Y. (2014). Activation of NLRP3 inflammasome by crystalline structures via cell surface contact. Scientific Reports, 4, 7281.

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Immunocytochemistry of Sarcomeric Actinin in Cells

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The cells were fixed in 4 % paraformaldehyde (PFA) and 0.05% Triton-X for 10 min. After fixation, the samples were incubated with a primary antibody against sarcomeric α-actinin (Sigma-Aldrich, MO, USA) and 4n,6-diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes, Thermo Fischer Scientific, MA, USA) for 2 h at room temperature, followed by a secondary antibody against rabbit IgG (H+L) conjugated to Alexa Fluor® 546 (Invitrogen, Thermo Fischer Scientific, MA, USA) for 1 h at room temperature. The samples were washed by 1X phosphate buffered saline (PBS, Gibco, Thermo Fischer Scientific, MA, USA) and were mounted on a glass slide with Prolong Gold anti-fade agent (Invitrogen, Thermo Fischer Scientific, MA, USA). Confocal microscopy (Zeiss LSM 7 LIVE) was used to obtain images of immunostained cells. 3D reconstruction of z-stacked images was performed using Zen lite 2.3 software (Zeiss).

Ahn S., Ardoña H.A., Lind J.U., Eweje F., Kim S.L., Gonzalez G.M., Liu Q., Zimmerman J.F., Pyrgiotakis G., Zhang Z., Beltran-Huarac J., Carpinone P., Moudgil B.M., Demokritou P, & Parker K.K. (2018). Mussel-inspired 3D Fiber Scaffolds for Heart-on-a-Chip Toxicity Studies of Engineered Nanomaterials. Analytical and bioanalytical chemistry, 410(24), 6141-6154.

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Immunofluorescence Analysis of Protein Localization

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For immunofluorescence analysis cells were seeded on glass cover slips, transfected with plasmids expressing indicated proteins and left for 24 h. Cells were fixed with 3.2% paraformaldehyde and permeabilised with 0.2% Triton X-100. The antibodies used for immunofluorescence analysis and their dilutions are presented in Supplementary Table 5.
Both primary and secondary antibodies were incubated for 30 min at room temperature. Nucleus coloration was obtained using Hoechst stain that was incubated alongside with secondary antibodies. After mounting with ProLong Gold anti-fade agent (Invitrogen, Molecular Probes Ref. P36930), cells were visualized using either Leica confocal TCS SP5 microscope with LAS-AF software for confocal imaging or Zeiss Axio Imager Z2 with scMOS ZYLA 5.5 camera and 40 × 1.3 NA magnification. 3D reconstructions from confocal Z-stacks were obtained using Imaris software. No fewer than 25 cells per sample in each experiment were analysed.

Arsic N., Slatter T., Gadea G., Villain E., Fournet A., Kazantseva M., Allemand F., Sibille N., Seveno M., de Rossi S., Mehta S., Urbach S., Bourdon J.C., Bernado P., Kajava A.V., Braithwaite A, & Roux P. (2021). Δ133p53β isoform pro-invasive activity is regulated through an aggregation-dependent mechanism in cancer cells. Nature Communications, 12, 5463.

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Immunofluorescence Staining of Cultured Cells

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For ICC, cells were plated on eight-well chamber slides coated with Matrigel and laminin (5 μg/mL). After 1 day, cells were washed once with PBS without calcium and magnesium (PBS - Ca²⁺/Mg²⁺) and fixed in 4% paraformaldehyde for 20 min, followed by washing with PBS + Ca²⁺/Mg²⁺. Cells were blocked with blocking buffer (10% donkey serum, 1% BSA, and 0.1% TritonX-100 in PBS + Ca²⁺/Mg²⁺) for at least 1 h and incubated with primary antibodies overnight (Additional file 1: Table S1) in antibody dilution buffer (1% donkey serum, 1% BSA, and 0.1% TritonX-100 in PBS + Ca²⁺/Mg²⁺). After overnight incubation, cells were washed three times with wash buffer (0.1% Triton X-100 in PBS + Ca²⁺/Mg²⁺). Cells were incubated with corresponding secondary antibodies (Additional file 1: Table S1), along with DAPI (1 mg/mL; ThermoFisher Scientific), diluted in antibody dilution buffer for 1 h. Following secondary antibody incubation, cells were washed twice with wash buffer and once with PBS + Ca²⁺/Mg²⁺. Slides were mounted with Prolong Gold anti-fade agent (Life Technologies). Images were obtained using a spinning-disk confocal microscope (Quorum) with MetaMorph software and were processed using ImageJ. For immunoblotting, cell lysate was extracted and 30 μg of protein was used per lane. Antibodies used are listed in Additional file 1: Table S1.

Lewis E.M., Meganathan K., Baldridge D., Gontarz P., Zhang B., Bonni A., Constantino J.N, & Kroll K.L. (2019). Cellular and molecular characterization of multiplex autism in human induced pluripotent stem cell-derived neurons. Molecular Autism, 10, 51.

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Immunofluorescent Staining of Cell-Cell Junctions

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Cells were fixed in 4 % paraformaldehyde (PFA) for 10 min, followed by permeabilization with 0.05% Triton-X for another 15 min. Non-specific binding was prevented by blocking with a 5% bovine serum albumin (BSA) solution in PBS for 1 h. After these steps, the samples were incubated with primary antibodies against vinculin (1:500; Abcam, MA, USA), VE-cadherin (1:500; Abcam, MA, USA), or fibronectin (1:500; Sigma, MO, USA) for 2 h at room temperature, followed by incubation with the appropriate secondary antibodies (against rabbit IgG (H+L) conjugated to Alexa Fluor 488 or mouse IgG conjugated to Alexa Fluor 633; 1:200; Life Technologies, CA, USA) and Alexa Fluor 546 Phalloidin (1:200; Life Technologies, CA, USA) for 1 h at room temperature. To stain for nuclei, samples were incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen, CA, USA) solution with a working concentration of at least 300 nM for 5 minutes. The samples were washed at least three times with 0.5% BSA solution in between steps. After the last staining step, all samples were washed with PBS for at least three times and then mounted on a glass slide with Prolong Gold anti-fade agent (Life Technologies, CA, USA).

Eweje F., Ardoña H.A., Zimmerman J.F., O’Connor B.B., Ahn S., Grevesse T., Rivera K.N., Bitounis D., Demokritou P, & Parker K.K. (2019). Quantifying the effects of engineered nanomaterials on endothelial cell architecture and vascular barrier integrity using a cell pair model. Nanoscale, 11(38), 17878-17893.

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