Prolong gold

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom, Japan, Canada, France, Spain, Australia

ProLong Gold is a mounting medium designed for preserving and protecting fluorescent signals in microscopy samples. It is a water-based, glycerol-based formulation that helps to reduce photobleaching and maintain the integrity of fluorescent proteins and dyes.

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Lab products found in correlation

1 520 protocols using prolong gold

Immunofluorescence Staining of DNA Damage Markers

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Cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized for 10 min with 0.2% Triton X-100. Cells were blocked for 1 hr in PBS containing either 3% BSA (Rad51), 1% normal goat serum (53BP1, H2AX) or 3% normal goat serum (H2AX for primary lung fibroblasts) and incubated with primary antibodies diluted in the respective blocking solution for 1 hr at room temperature. Antibodies used were: rabbit anti-Rad51 (Calbiochem, #PC130, 1:100), rabbit anti-53BP1 (a gift from Zhenkun Lou, 1:500), rabbit anti-γH2AX (Cell Signaling Technology, #2577, 1:500). After washing with PBS, cells were stained with secondary antibodies (Invitrogen, Alexa fluor 488 goat anti-rabbit IgG, #A11034, 1:2,000) diluted in the blocking solution for 30 min at room temperature. Cells were washed with PBS, stained with DAPI (0.1 μg ml⁻¹, Sigma) for 5 min and mounted with ProLong Gold (Invitrogen). For analyses of micronuclei and chromatin bridges, cells were fixed as above or with methanol:acetone (3:1) for 10 min at −20°C and permeabilized for 10 min with 0.2% Triton X-100 at room temperature, stained with DAPI (0.1 μg ml⁻¹, Sigma) for 5 min and mounted with ProLong Gold (Invitrogen). Slides were viewed and photographed on a Zeiss fluorescent microscope with a 100× objective.

Maskey R.S., Kim M.S., Baker D.J., Childs B., Malureanu L.A., Jeganathan K.B., Machida Y., van Deursen J.M, & Machida Y.J. (2014). Spartan deficiency causes genomic instability and progeroid phenotypes. Nature Communications, 5, 5744.

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Immunofluorescent Staining of Smooth Muscle Cells

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OCT was removed from sections by 3, 5 minute washes in Tris buffered saline (TBS; 100 mM Tris pH7.6, 150 mM NaCl). Slides were mounted in Prolong Gold containing DAPI (Invitrogen) and visualized by confocal microscopy (Olympus Fluoview FV1000). For immuno-fluorescent staining cryosections were permeabilized in 0.2% triton in TBS for 5 minutes, washed in TBS for 5 minutes them blocked in 5% goat serum diluted in TBS at room temperature for 1 hour. Blocked sections were incubated for 4-5 hours at 37° with primary antibodies to, the SM2 isoform of smooth muscle myosin heavy chain (1:500)
[11 (link)], CD31 (1:100, clone 390, Affymetrix, eBioscience), CD68 (1:200, clone FA-11, AbD Serotec) diluted in 5% goat serum/TBS. Some sections were incubated without primary antibody as a negative control. Following washing in TBS primary antibodies were detected by incubation with anti-rabbit or anti-rat Alexa Fluor 647 (1:10,000, Jackson ImmunoResearch). After washing slides were mounted in Prolong Gold containing DAPI (Invitrogen) and visualized by confocal microscopy.

Herring B.P., Hoggatt A.M., Burlak C, & Offermanns S. (2014). Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury. Vascular Cell, 6, 21.

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Bacterial Imaging and Cellular Analysis

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Bacteria from in vitro culture medium were sonicated and formaldehyde fixed as described above, then washed with PBS and mounted onto clean microscope slides using Prolong Gold (Invitrogen). Similarly, infected macrophages on coverslips were fixed with 4 % formaldehyde for 30 min, washed with PBS and mounted onto clean microscope slides using Prolong Gold (Invitrogen). Cells were observed using a confocal microscope (LSM 780; Carl Zeiss) equipped with a GaAsp detector, using a plan-apochromat ×63/1.4 oil DIC M27 objective. Samples were excited with a 488 and 561 nm laser, using 490–516 nm and 585–696 nm filters for green and red fluorescence, respectively. Transmitted light images for infected macrophages were collected using a confocal microscope equipped with a transmitted light detector. Images were acquired through z-stack acquisition, with an increment of 0.500 µm between image frames, and displayed as maximum-intensity projections. Zen imaging software (Zen SP1 2012, Black edition, version 8.1.0.484) was used to view and process images.

Mouton J.M., Helaine S., Holden D.W, & Sampson S.L. (2016). Elucidating population-wide mycobacterial replication dynamics at the single-cell level. Microbiology, 162(6), 966-978.

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Immunostaining of Oocytes and Embryos

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Immunostaining of late stage oocytes was carried out as described [8] (link) under conditions to limit activation. For experiments enriching for prometaphase I oocytes, females were yeasted for 2–3 days with males. For preparations enriching for metaphase I oocytes, virgin females were yeasted for 4–5 days post-eclosion [7] . Ovaries were treated with the primary and secondary antibodies described below and with 1.0 µg/mL 4′6-diamididino-2-phenylindole (DAPI) to label the DNA and mounted in ProLong Gold (Invitrogen). Immunostaining of embryos was carried out as described [50] (link). The DNA was labeled with 2.5 µg/mL Hoechst 34580 (Invitrogen) and mounted in ProLong Gold (Invitrogen).
Primary antibodies were used at the following concentrations: rat anti-α-tubulin (AbD Serotec, NC 1∶250), mouse anti-α-tubulin DM1a (Sigma-Aldrich 1∶100), rat anti-CID [51] (link) 1∶1000), rabbit anti-trimethylated-histone-3 at lysine 9 (AbCam 1∶250) and rabbit anti-phosphorylated-histone 3 at serine 10 (Millipore 3∶1000). Secondary Alexa-488, Alexa-555, or Alexa-647 conjugated antibodies (Molecular Probes) were used at a dilution of 1∶400.

Hughes S.E, & Hawley R.S. (2014). Topoisomerase II Is Required for the Proper Separation of Heterochromatic Regions during Drosophila melanogaster Female Meiosis. PLoS Genetics, 10(10), e1004650.

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Lysotracker and Lamp1 Imaging in iGFP-NLSm Cells

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iGFP-NLSm stable cells were plated onto coverslips, transfected with shTmem106b or control, and induced with doxycycline as described above. 72 hours after transfection, cells were treated with Lysotracker 594 (Invitrogen) at a dilution of 1:1000 in standard media and incubated at 37 degrees C for 30 minutes. After this incubation, cells were fixed for fifteen minutes in 2% paraformaldehyde, washed 4 times with PBS and then incubated with Draq5 (Invitrogen) for 30 minutes. The coverslips were then placed onto slides using mounting media (ProLong Gold, Invitrogen). Lamp1 was stained by fixing cells using 2% paraformaldehyde, washing with PBS, and blocking using 3% BSA, 0.05% saponin for one hour. Lamp1 antibody H4A3 (DSHB) was diluted into the blocking buffer at 1 μg/mL and allowed to incubate overnight at 4 degrees C in a humid chamber. Alexa 594 (Invitrogen) secondary was used at a dilution of 1:1000 and incubated for one hour. Cells were then washed and incubated with Draq5 for 30 minutes, then mounted onto slides (ProLong Gold, Invitrogen). Images were captured using a Leica SP5 confocal microscope.

Mao F., Robinson J., Unger T., Posavi M., Amado D., Elman L., Grossman M., Wolk D., Lee E., Van Deerlin V.M., Porta S., Lee V., Trojanowski J, & Chen-Plotkin A.S. (2021). TMEM106B modifies TDP-43 pathology in human ALS brain and cell-based models of TDP-43 proteinopathy. Acta neuropathologica, 142(4), 629-642.

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Immunofluorescence Staining of DNA Repair Markers

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Cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized for 10 min with 0.2% Triton X-100. Cells were blocked for 1 h in PBS containing 3% BSA (Rad51), 1% normal goat serum (53BP1, γH2AX) or 3% normal goat serum (γH2AX for primary lung fibroblasts) and incubated with primary antibodies diluted in the respective blocking solution for 1 h at room temperature. Antibodies used were: rabbit anti-Rad51 (Calbiochem, #PC130, 1:100), rabbit anti-53BP1 (a gift from Zhenkun Lou, 1:500) and rabbit anti-γH2AX (Cell Signaling Technology, #2577, 1:500). After washing with PBS, cells were stained with secondary antibodies (Invitrogen, Alexa fluor 488 goat anti-rabbit IgG, #A11034, 1:2,000) diluted in the blocking solution for 30 min at room temperature. Cells were washed with PBS, stained with DAPI (0.1 μg ml⁻¹, Sigma) for 5 min and mounted with ProLong Gold (Invitrogen). For analyses of micronuclei and chromatin bridges, cells were fixed as above or with methanol:acetone (3:1) for 10 min at −20 °C and permeabilized for 10 min with 0.2% Triton X-100 at room temperature, stained with DAPI (0.1 μg ml⁻¹, Sigma) for 5 min and mounted with ProLong Gold (Invitrogen). Slides were viewed and photographed on a Zeiss fluorescent microscope with a × 100 objective.

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Immunofluorescence Staining Protocol

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Tissues were first permeabilized for 10 min with 0.2% Triton-X (OmniPur) diluted in phosphate buffered saline (PBS) (first base). The slides were then blocked with 3% horse serum (Invitrogen) in 0.1% Triton-X (OmniPur) in PBS. Primary antibodies were diluted in blocking buffer and added onto the slides before incubating them overnight at 4 °C. The following day, corresponding secondary antibodies were diluted 1:1000 each and incubated for 2 h at room temperature. Freshly prepared 0.1 μg/mL DAPI (Sigma) was prepared and the slides were incubated for 10 min and mounted with Prolong Gold (molecular probes). The mounting agent was allowed to set overnight away from light before subsequent confocal imaging.

Kamath S.P, & Chen A.I. (2018). Myocyte Enhancer Factor 2c Regulates Dendritic Complexity and Connectivity of Cerebellar Purkinje Cells. Molecular Neurobiology, 56(6), 4102-4119.

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Quantifying Microglial Activation in Transgenic Mice

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To confirm Iba-1 co-labeling with GFP expression in CX3CR1-GFP mice, V1 and Cb coronal sections underwent Iba-1 immunohistochemistry as described in (Sipe et al. 2016 , Lowery et al. 2017 (link)). Briefly, V1 and Cb sections were rinsed in RT 0.1M PBS (three 10 minute washes) and incubated in a methanol/hydrogen peroxide block to quench endogenous peroxidases and blocked in bovine serum albumen (BSA). Sections were incubated in primary antibody solution for 48hrs at 4°C on a shaker in a humidified chamber (anti-IBA-1 1:2500, Wako 019–19741). The sections were then washed with 0.1M PBS and left for 4 hours at RT in secondary antibody (Alexa-Fluor 594 1:500, donkey anti-rabbit; Invitrogen A21207).
For qualitative evaluation of P2RY12 expression, sections were incubated with primary antibody (rabbit-anti-P2RY12 1:2000, Anaspec AS-55043A) overnight at 4ºC (in PBS containing 0.3% Triton-X and 0.5% BSA). Sections were washed in PBS and incubated with secondary antibody (Alexa-Fluor 594 1:500, goat anti-rabbit; Invitrogen A11012). Sections were mounted onto glass slides with coverslips using Prolong Gold (Molecular Probes, P36934).

Stowell R.D., Wong E.L., Batchelor H.N., Mendes M.S., Lamantia C.E., Whitelaw B.S, & Majewska A.K. (2018). Cerebellar microglia are dynamically unique and survey Purkinje neurons in vivo. Developmental neurobiology, 78(6), 627-644.

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Immunofluorescent Labeling of Inner Ear Sensory Organs

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Inner ears were fixed for 2 hours in a solution of 4% paraformaldehyde prepared in 67mM Sorenson’s phosphate buffer (pH 7.4). For immunofluorescent labeling, inner ear sensory organs were micro-dissected to expose the sensory epithelia and permeabilized and blocked using blocking solution (5% donkey serum, 1% BSA, and PBS) supplemented with Triton X-100 to 0.5%. Primary antibodies and phalloidin Alexa Fluor 488 (Invitrogen A12379) were diluted in blocking solution supplemented with Tween-20 to 0.1% and incubated with the tissue overnight at 4°C. Tissue was washed thoroughly with PBS-T (PBS and 0.05% Tween-20), followed by incubation with species-specific, Alexa Fluor-conjugated (Invitrogen) or DyLight-conjugated (Jackson ImmunoResearch) secondary antibodies. Tissue was subsequently washed with PBS-T, mounted using Prolong Gold (Molecular Probes, P36930), and imaged via structured illumination microscopy using a Zeiss Axio Imager M.2 with ApoTome.2 attachment. Images were collected with Zeiss Zen software, and figures were prepared with Adobe Illustrator. The following commercial antibodies were used in this study: β2-Spectrin (BD Biosciences 612562); Oncomodulin (Santa Cruz Sc7446). The Celsr1 antibody was provided by D. Devenport and has been previously described (Devenport and Fuchs, 2008 (link)).

Duncan J.S., Stoller M.L., Francl A.F., Tissir F., Devenport D, & Deans M.R. (2017). Celsr1 Coordinates the Planar Polarity of Vestibular Hair Cells During Inner Ear Development. Developmental biology, 423(2), 126-137.

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Immunofluorescence Staining of Tetrahymena

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Cells were washed in 10 mM Tris, pH 7.4, and then fixed in 3.2% paraformaldehyde and 0.25% Triton X-100 in PHEM buffer (60 mM 1,4-piperazinediethanesulfonic acid, 25 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid, 10 mM EGTA, and 2 mM MgCl₂, pH 6.9) for 10 min at 23°C. Cells were washed twice in 0.1% BSA-PBS, and either dried on a coverslip at 23°C or maintained in Eppendorf tubes for the remainder of the staining protocol. Blocking was performed in 1% BSA-PBS for 20 min, followed by incubation in primary antibody diluted in 1% BSA-PBS for 1 h at 23°C. Primary antibodies used in this study were mouse anti-Kl Ag (1:100; Williams et al., 1990 (link)), rabbit anti-TtCen1 (1:2,000; Stemm-Wolf et al., 2005 (link)), mouse anti-α-tubulin (1:200, 12G10, Developmental Studies Hybridoma Bank, University of Iowa; Wloga et al., 2010 (link)), and rabbit anti-TtSas6A (1:250; Culver et al., 2009 (link)). Cells were then washed twice with 0.1% BSA-PBS before incubation with secondary antibodies diluted in 1% BSA-PBS. Secondary antibodies used in this study were IgG derived from goat and conjugated to Alexa Fluor 488, 594, or 647 (Invitrogen) and diluted to 1:1,000. Hoechst 33342 DNA dye (Sigma-Aldrich) was used at 1:1,000. Cells were mounted in either Citifluor (Ted Pella) or Prolong Gold (Molecular Probes) mounting media. Coverslips were sealed with clear nail polish.

Ruehle M.D., Stemm-Wolf A.J, & Pearson C.G. (2020). Sas4 links basal bodies to cell division via Hippo signaling. The Journal of Cell Biology, 219(8), e201906183.

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