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Prolong gold reagent

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ProLong Gold reagent is a mounting medium used for fluorescence microscopy. It is designed to enhance and preserve fluorescent signals in fixed and permeabilized samples.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (9 mentions) Matlab, by MathWorks (7 mentions) Triton x 100, by Merck Group (4 mentions) Axioscope a1, by Thermo Fisher Scientific (3 mentions) Zen software, by Zeiss (3 mentions) Hoechst 33342, by Thermo Fisher Scientific (3 mentions) Af488 labeled secondary goat ant rabbit immunoglobulin g antibody, by Thermo Fisher Scientific (3 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Fv1000 spectral confocal microscope, by Olympus (2 mentions) Rabbit anti mouse lamp1 primary antibody, by Abcam (2 mentions) Donkey anti rabbit alexafluor 647 secondary antibody, by Thermo Fisher Scientific (2 mentions) Alexa fluor 546 phalloidin, by Thermo Fisher Scientific (2 mentions) Dmi3000, by Leica (2 mentions) Dfc365fx, by Leica (2 mentions) Slidebook software, by Olympus (2 mentions) Spinning disc confocal microscope dsu ix81, by Olympus (2 mentions) Axiovision 40v 4, by Zeiss (2 mentions) Blocking one reagent, by Nacalai Tesque (2 mentions)

Close spelling variants of this product

ProLong Gold antifade reagent (2699 citations) ProLong Gold (1530 citations) Prolong Gold antifade mounting reagent (50 citations) Prolong Gold mounting reagent (22 citations) Prolong Gold Antifading reagent (19 citations) Prolong Gold Antifade mounting reagent with DAPI (12 citations) DAPI-Prolong Gold anti-fade reagent (11 citations) ProLong Gold antifade reagent mounting medium (8 citations) Prolong gold DAPI antifade reagent (7 citations) ProLong Gold mounting reagent with DAPI (6 citations)

65 protocols using prolong gold reagent

1

Quantitative Analysis of cMet Expression

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The cells were blocked with 1X PBS plus 2% BSA for 1 hour at 4 °C, and were incubated with 5 μM of peptide for 10 min at room temperature (RT) in the dark, washed 3×, and fixed with 4% PFA for 5 min, washed with 1X PBS, and then mounted on glass slides with ProLong Gold reagent containing DAPI (Invitrogen). A 1:3000 dilution of primary monoclonal rabbit anti-cMet antibody (#8198, Cell Signaling Technology) was incubated with the cells in vitro as a positive control. Afterwards, the cells were incubated with a 1:500 dilution of AF488-labeled secondary goat ant-rabbit immunoglobulin G antibody (#A-11029, Life Technologies), and then mounted on glass slides with ProLong Gold reagent containing DAPI. Confocal fluorescence images were collected using a 63X oil-immersion objective (Leica SP5 Inverted 2-Photon FLIM confocal). Fluorescence intensities were quantified using custom Matlab software (Mathworks).
Wu X., Zhou J., Wang F., Meng X., Chen J., Chang T.S., Lee M., Li G., Li X., Appelman H.D., Kuick R, & Wang T.D. (2019). Detection of colonic neoplasia in vivo using near-infrared-labeled peptide targeting cMet. Scientific Reports, 9, 17917.
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2

LiLa-mediated Lysosomal Dynamics Imaging

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Cells were seeded on glass coverslips 24 h before use. After treatment with 2 μL/mL of LiLa, the cells were washed three times with PBS, fixed with 4% formaldehyde for 10 min, and then permeabilized with 0.2% Triton X-100 for 5 min. Filamentous actin was labeled with AlexaFluor-546 phalloidin (Invitrogen) for 30 min according to manufacturer's instructions. For LAMP1 immunostaining, the cells were treated with various LiLa as mentioned above and in the text, fixed, permeabilized, blocked with 5% BSA in PBS for 1 h at room temperature, and incubated with rabbit anti-mouse LAMP1 primary antibody (Abcam) overnight at 4 °C. After washing extensively with PBS, the cells were incubated for 2 h at room temperature with donkey anti-rabbit AlexaFluor 647 secondary antibody (Life Technologies). All secondary antibody incubations were performed in the dark to minimize photo bleaching. The cells were then washed extensively in PBS, stained with DAPI (Life Technologies) for 10 min, and mounted on glass slides with Prolong Gold reagent (Life Technologies). The stained cells were imaged on an Olympus FV1000 spectral confocal microscope using 60× objective.
Bagalkot V., Badgeley M.A., Kampfrath T., Deiuliis J.A., Rajagopalan S, & Maiseyeu A. (2015). Hybrid nanoparticles improve targeting to inflammatory macrophages through phagocytic signals. Journal of controlled release : official journal of the Controlled Release Society, 217, 243-255.
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3

Immunofluorescence Staining of Cellular Proteins

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Paraformaldehyde-fixed cells were permeabilized with 0.3% TritonX-100 in PBS and blocked with 3% FBS in PBST for 30 min. They were then incubated with anti-YAP antibody at 4 °C overnight and washed with PBS three times. Cells were incubated with anti-rabbit IgG-Alexa Fluor 488 for 1 h at room temperature, and washed with PBS three times. For confocal microscopy, cells were mounted in Prolong Gold reagent containing 10 μg/ml Hoechst 33342 (Life Technologies). When appropriate, cells were stained with phalloidin-Alexa Fluor 594 (Life Technologies) prior to mounting. Images were obtained with an FV1000-D confocal microscope equipped with a 40× objective lens using FV10-ASW software (Olympus). For screening of small molecules, PBS containing 10 μg/ml Hoechst 33342 was added and images were obtained by IN Cell Analyzer 2000 (GE Healthcare) using a 40× objective lens.
Oku Y., Nishiya N., Shito T., Yamamoto R., Yamamoto Y., Oyama C, & Uehara Y. (2015). Small molecules inhibiting the nuclear localization of YAP/TAZ for chemotherapeutics and chemosensitizers against breast cancers. FEBS Open Bio, 5, 542-549.
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4

EdU Click-iT Assay for DNA Replication

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For DNA replication analysis, we used the Click-iT EdU Alexa Fluor 488 Kit (#C10337; Life Technologies) according to the manufacturer’s instruction. Briefly, EdU was added to the culture medium of hPSCs or fibroblasts grown on top of coverslips to a final concentration of 10 µM EdU and incubated for 1 h in the presence of 5% O2, 5% CO2 at 37°C. Then, cells were immediately washed twice with DPBS 1X and fixed with PFA 4% in DPBS 1X for 10 min at room temperature. Fixed samples were then washed twice with 3% BSA in DPBS 1X, permeabilized with 0.2% Triton X-100 0.2% for 20 min at room temperature, and washed twice with 3% BSA in DPBS 1X, and the cocktail reaction (prepared according to the manufacturer’s instruction) was added to the samples. Samples were incubated for 30 min protected from light, and then immediately washed twice with 3% BSA in DPBS 1X. The cover glass was lowered on top of a 6-µl drop of ProLong Gold reagent (#P-36931; Life Technologies) placed on top of a microscope slide (#2949-75X25; Corning). Cells were imaged 4–24 h after mounting using a Leica DM6B upright digital research microscope (Leica Microsystems).
Vessoni A.T., Zhang T., Quinet A., Jeong H.C., Munroe M., Wood M., Tedone E., Vindigni A., Shay J.W., Greenberg R.A, & Batista L.F. (2021). Telomere erosion in human pluripotent stem cells leads to ATR-mediated mitotic catastrophe. The Journal of Cell Biology, 220(6), e202011014.
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5

Immunohistochemical Analysis of Lung Tissue

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At day 30 after x31 infection, lung tissues were harvested, embedded in OCT compound (Sakura Finetek), and frozen in liquid nitrogen. 6-µm-thick cryosections were prepared and fixed for 10 min with acetone. Fixed sections were rehydrated with PBS, blocked with Blocking One reagent (Nacalai Tesque), and stained with a mixture of Abs against CD45 (30-F11; BD Biosciences), Pan-Cytokeratin (rabbit polyclonal; Bioss), and either CXCL16 (goat polyclonal; R&D Systems) or control Abs (goat IgG; Jackson Immunoresearch) dissolved in PBS containing 2% BSA for 30 min at 37°C. Sections were further incubated with Alexa Fluor–labeled appropriate secondary Abs. After the staining, sections were mounted with Prolong Gold reagent (Life Technologies) and then photographed using an SP5 confocal microscope (Leica Microsystems).
Takamura S., Kato S., Motozono C., Shimaoka T., Ueha S., Matsuo K., Miyauchi K., Masumoto T., Katsushima A., Nakayama T., Tomura M., Matsushima K., Kubo M, & Miyazawa M. (2019). Interstitial-resident memory CD8+ T cells sustain frontline epithelial memory in the lung. The Journal of Experimental Medicine, 216(12), 2736-2747.
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6

Fibroblast Lysosome and Cholesterol Imaging

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Fibroblasts were seeded in round gelatin-coated glass coverslips at a density of 7,400 cells/cm2. To label the lysosomes, fibroblasts were incubated with 70 nM lysoTracker Red DND-99 probe (Invitrogen, Madrid, Spain; λem = 590 nm) for 30 min at 37°C. Next, fibroblasts were fixed with 3% paraformaldehyde (PFA) for 30 min at RT, washed with glycine and stained with 25 μg/ml Filipin (Sigma; λem = 400–484 nm) for free cholesterol detection. Finally, the round covers were mounted with Prolong® Gold reagent (Life Technologies, Alcobendas, Spain) and observed using the fluorescence microscope (Leica, Wetzlar, Germany).
Costa-Laparra I., Juárez-Escoto E., Vicario C., Moratalla R, & García-Sanz P. (2023). APOE ε4 allele, along with G206D-PSEN1 mutation, alters mitochondrial networks and their degradation in Alzheimer’s disease. Frontiers in Aging Neuroscience, 15, 1087072.
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7

Immunofluorescence Staining and Imaging

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Immunofluorescence staining was performed as previously described (Pagan et al., 2003 (link)). Cells were permeabilized using 0.1% Triton X-100 for 5 min, then incubated with primary and secondary antibodies. Coverslips were mounted in ProLong Gold reagent (Life Technologies) before imaging. For live-cell and fixed-cell imaging experiments, RAW 264.7 macrophages were cultured on glass-bottom, 35-mm dishes (MatTek) and in 24-well culture dishes (Thermo Scientific) with coverslips, respectively. Epifluorescence still images were taken with a 12-Mp differential contrast Roper CoolSNAP HQ2 monochrome camera (DP71; Olympus) using a Personal Deltavision Olympus IX71 inverted wide-field deconvolution microscope fitted for a 60×/numerical aperture (NA) 1.35 oil objective using the associated DPController software (version 2.1; Olympus). Imaging Z-stacks of fixed cells was set at 0.2 μm while imaging of live cells was set at 0.3 μm Z-stack and a 15 s time-lapse interval for 20 min.
Yeo J.C., Wall A.A., Luo L, & Stow J.L. (2015). Rab31 and APPL2 enhance FcγR-mediated phagocytosis through PI3K/Akt signaling in macrophages. Molecular Biology of the Cell, 26(5), 952-965.
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8

Multiplex FISH Analysis of A-673 Cells

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A-673 cells were cultured on poly-lysine coated #1 coverslips (Neuvitro) in DMEM (Life Technologies) supplemented with 10% FBS (Sigma) and 1% Pen Strep (Life Technologies). Cells were treated with either DMSO, 10 μM compound 1, or 10 μM compound 3 for 4 h before fixation with 4% paraformaldehyde (Electron Microscopy Sciences) in PBSM (1X PBS with 5mM MgCl2) for 20 min at room temperature. Cells were washed two times with PBSM and permeabilized with 0.5% (v/v) Triton X-100 (Sigma) in PBSM for 5 min at room temperature. After permeabilization, cells were washed two times with PBSM and then incubated with pre-hybridization solution (2X SCC, 10% (v/v) formamide (Sigma)) for 5 min at room temperature. Cells were then hybridized with 250nM of NKX2-2 or RUNX3 FISH probes fluorescently labeled with Quasar 570 (Biosearch Technologies) in 2X SSC, 10% (v/v) formamide (Life Technologies), 10% (w/v) dextran sulfate (Sigma) for 16 h at 37°C. Following hybridization, cells were washed two times with pre-hybridization solution warmed to 37°C for 30 min. Cells were washed three times with PBSM for 5 min at room temperature before counterstaining DNA with DAPI (0.5 mg l-1) and mounted on slides using ProLong gold reagent (Life Technologies). For sequences of FISH probes, see Supplementary Table 4.
Ross N.T., Lohmann F., Carbonneau S., Fazal A., Weihofen W.A., Gleim S., Salcius M., Sigoillot F., Henault M., Carl S.H., Rodríguez-Molina J.B., Miller H.R., Brittain S.M., Murphy J., Zambrowski M., Boynton G., Wang Y., Chen A., Molind G.J., Wilbertz J.H., Artus-Revel C.G., Jia M., Akinjiyan F.A., Turner J., Knehr J., Carbone W., Schuierer S., Reece-Hoyes J.S., Xie K., Saran C., Williams E.T., Roma G., Spencer M., Jenkins J., George E.L., Thomas J.R., Michaud G., Schirle M., Tallarico J., Passmore L.A., Chao J.A, & Beckwith R.E. (2019). CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing’s sarcoma. Nature chemical biology, 16(1), 50-59.
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9

Immunocytochemical Analysis of Bovine OECs

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Primary bovine OECs were grown on coverslips coated with 0.2% gelatin and fixed with 4% formaldehyde. Cells were then permeabilized with 0.1% Triton X-100 in PBS for 1 minute and blocked using 5% normal goat serum for 30 minutes. Coverslips were subsequently incubated with a mouse monoclonal anti-cytokeratin antibody (1:200 dilution; Cell Signaling Technology) for 1 hour. Coverslips were then washed three times using PBS and incubated with Alexa Fluor conjugated anti-mouse Fab’ fragments for 30 minutes, washed again with PBS, counterstained/mounted with 4’,6-diamidino-2-phenylindole (DAPI) containing Prolong Gold reagent (Life Technologies, Carlsbad, CA). Images were acquired using an inverted microscope (DMI 3000, Leica) using a cooled monochromatic camera (DFC365FX, Leica).
Pillai V.V., Weber D.M., Phinney B.S, & Selvaraj V. (2017). Profiling of proteins secreted in the bovine oviduct reveals diverse functions of this luminal microenvironment. PLoS ONE, 12(11), e0188105.
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10

Characterization of Bovine iPSCs

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Bovine iPSCs were grown on coverslips seeded with irradiated MEF feeders and fixed with 4% formaldehyde. Cells were then permeabilized with 0.1% Triton X-100 in PBS for 1 min and blocked using 5% normal goat serum for 30 min. Coverslips were subsequently incubated with antibodies against SSEA1, 4 and 3 (1:200 dilution; Iowa Hybridoma Bank) for 1 h. Coverslips were then washed three times using PBS, incubated with Alexa Fluor-conjugated anti-mouse Fab’ fragments for 30 min, washed again with PBS, and counterstained/mounted with 4′,6-diamidino-2-phenylindole (DAPI) containing Prolong Gold reagent (Life Technologies, Carlsbad, CA, USA). Images were acquired using an inverted microscope (DMI 3000, Leica) using a cooled monochromatic camera (DFC365FX, Leica).
Pillai V.V., Koganti P.P., Kei T.G., Gurung S., Butler W.R, & Selvaraj V. (2021). Efficient induction and sustenance of pluripotent stem cells from bovine somatic cells. Biology Open, 10(10), bio058756.
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