Prolong diamond antifade mountant with dapi

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ProLong Diamond Antifade Mountant with DAPI is a laboratory product designed for use in fluorescence microscopy. It is a mounting medium that helps preserve fluorescent signals and prevent photobleaching. The product contains the nuclear stain DAPI, which binds to DNA and emits blue fluorescence.

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542 protocols using prolong diamond antifade mountant with dapi

Immunofluorescence Staining for Viral Infections

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For CVB3 infections, after fixation, the cells were permeabilized with 0.1% saponin in 1x PBS containing 3% BSA at 4°C for 20 min and blocked in 1x PBS containing 3% BSA at RT for 1 h. Then the cells were incubated with VP1 primary antibody (EMD Millipore, Cat#MAB948, 1:1000) or anti-dsRNA antibody (J2, 1:1,000) in 1x PBS containing 1% BSA for 1h at RT. Cell were washed five time with PBS, incubated with 1:5,000 Alexa Fluor-488 secondary antibodies (Invitrogen) in 1x PBS containing 1% BSA for 1h at room temperature in the dark, followed by five washes with PBS, and staining with ProLong^™ Diamond Antifade Mountant with DAPI (Invitrogen, Cat#P36962).
For confocal immunofluorescence studies, the indicated antibodies and dyes were diluted as below: mouse anti-Flag (Sigma, M2, 1:1,000), rabbit anti-HA (Pierce, 1:1,000), goat-anti-mouse Alexa Fluor-488 (Invitrogen, 1:1,000), goat-anti-rabbit Alexa Fluor-488 (Invitrogen, 1:1,000), goat-anti-mouse Alexa Fluor-555 (Invitrogen, 1:1,000), goat-anti-rabbit Alexa Fluor-647 (Invitrogen, 1:1,000), Bodipy493/503 (ThermoFisher, Cat#3922, 15 μg/mL), ProLong^™ Diamond Antifade Mountant with DAPI.

Fan W., Mar K.B., Sari L., Gaszek I.K., Cheng Q., Evers B.M., Shelton J.M., Wight-Carter M., Siegwart D.J., Lin M.M, & Schoggins J.W. (2021). TRIM7 inhibits enterovirus replication and promotes emergence of a viral variant with increased pathogenicity. Cell, 184(13), 3410-3425.e17.

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Immunofluorescent Detection of γ-H2AX Foci

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Cells were grown in chamber slides and then treated with drug for 24 h. Paraformaldehyde was added to cells to a final concentration of 4%. Cells were permeabilized with 0.5% Triton X-100 for 10 min. Primary antibody to γ-H2AX was added to cells in 5% goat serum in PBS (blocking buffer) for 1 h. Cells were washed with PBS and incubated with fluorescein (FITC)-conjugated secondary antibody in blocking buffer for 1 h. Cells were mounted with ProLong Diamond Antifade Mountant with DAPI (Life Technologies, Grand Island, NY) nuclear stain and visualized using the Nikon Eclipse Ti fluorescent microscope. Cells positive for γ-H2AX foci were counted from four independent fields.

Dolfi S.C., Medina D.J., Kareddula A., Paratala B., Rose A., Dhami J., Chen S., Ganesan S., Mackay G., Vazquez A, & Hirshfield K.M. (2017). Riluzole exerts distinct antitumor effects from a metabotropic glutamate receptor 1-specific inhibitor on breast cancer cells. Oncotarget, 8(27), 44639-44653.

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Quantification of DM2 Nuclear Foci

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Fibroblasts were fixed with 4% w/v PFA, permeabilized using 1% Triton X-100 in DPBS for 20 min at RT, blocked with Background Sniper (Biocare Medical), incubated with rabbit anti-RBFOX2 primary antibody at 1:500 (Sigma Aldrich) overnight at 4°C, and then incubated with 1:1000 goat anti-rabbit AlexaFluor 647 (Thermo) for 1 hr at RT. Fibroblasts samples were re-fixed, washed in DPBS, pre-hybridized for 30 min at 37°C, and then probed for 4 hrs at 50°C with a Cy3-(CAG)₈ DM1 or Cy3-(CAGG)₆ DM2 probe (IDT). Slides were washed with 42°C pre-warmed 40% v/v formamide in 2X SSC and mounted using ProLong Diamond Antifade mountant with DAPI (Life Technologies). Fibroblasts were imaged on a Zeiss LSM 840 confocal scanning microscope with a 40X water objective. All images were taken at the same laser intensities and settings. In DM2 fibroblasts, the foci were counted blind in at least 100 nuclei per replicate (at least 300 nuclei over three replicates) for all furamidine treatments. The number of nuclear foci for each cell was quantified using Fiji.

Jenquin J.R., O’Brien A.P., Poukalov K., Lu Y., Frias J.A., Shorrock H.K., Richardson J.I., Mazdiyasni H., Yang H., Huigens RW I.I.I., Boykin D., Ranum L.P., Cleary J.D., Wang E.T, & Berglund J.A. (2022). Molecular characterization of myotonic dystrophy fibroblast cell lines for use in small molecule screening. iScience, 25(5), 104198.

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Immunofluorescent Colocalization of mTOR and LAMP-1

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As methodology reported before [6 (link)], cells were grown on coverslips and fixed with 3.7% formaldehyde for 15 min, followed by permeabilization using 0.1% Triton-X 100 for 10 min. The coverslips were washed 3 times using PBS for 5 min; blocked in 3% BSA for 30 min and incubated with primary antibody for 40 min at 37°C. Co-localization of mTOR and LAMP-1 was performed by mixing both primary antibodies and adding to the coverslips, and then incubated for 40 min at 37°C. The list of the primary antibodies used, along with their source, catalogue numbers and dilutions is provided in S9 Table. The coverslips were washed 3 times with PBS for 3 min each. The primary antibody was detected by incubating cells with species specific Alexa-Fluor 488 or Alexa-Fluor 568 secondary antibody for 30 min at 37 ^oC. The coverslips were washed 3 times with PBS and mounted with Prolong Diamond Antifade Mountant with DAPI (Life Technologies). The stained cells were observed and imaged using Olympus FV3000 scanning confocal microscope. Two coverslips per sample were set up and z-stack images of a minimum of 5 fields per coverslips were captured with 40x objective lens. Co-localization analysis of z-stack images was performed using Olympus cellSens software.

Shrestha S., Singhal S., Sens D.A., Somji S., Davis B.A., Guyer R., Breen S., Kalonick M, & Garrett S.H. (2021). Elevated glucose represses lysosomal and mTOR-related genes in renal epithelial cells composed of progenitor CD133+ cells. PLoS ONE, 16(3), e0248241.

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In Situ Hybridization of Adipose Tissue

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Inguinal white adipose tissue (iWAT) from CL-treated WT mice (Jackson Laboratory, #000664) was fixed in 10% formalin overnight, embedded with paraffin, and sectioned into unstained, 5-μm-thick sections. Sections were baked at 60°C for 1 hr, deparaffinized, and baked again at 60°C for another hour prior to pre-treatment. The standard pre-treatment protocol was followed for all sectioned tissues. In situ hybridization was performed according to manufacturers’ instructions using the RNAscope Multiplex Fluorescent Reagent Kit v2 (#323136, Advanced Cell Diagnostics [ACD], Newark, CA). Opal fluorophore reagent packs (Akoya Biosciences, Menlo Park, CA) for Opal 520 (FP1487001KT), Opal 570 (FP1488001KT), and Opal 620 (FP1495001KT) were used at a 1:1000 dilution in TSA buffer provided in the RNAscope Multiplex Fluorescent Reagent Kit v2. Probes targeting mm-Adrb3 (#495521, ACD) in channel 1, mm-Ucp1 (#455411-C2, ACD) in channel 2, and mm-Ppargc1b (#402131-C4, ACD) in channel four were used. Slides were mounted with ProLong Diamond Antifade Mountant with DAPI (P36966, Life Technologies). Fluorescent signals were captured with the 40x objective lens on a laser scanning confocal microscope (LSM880; Zeiss).

Rajbhandari P., Arneson D., Hart S.K., Ahn I.S., Diamante G., Santos L.C., Zaghari N., Feng A.C., Thomas B.J., Vergnes L., Lee S.D., Rajbhandari A.K., Reue K., Smale S.T., Yang X, & Tontonoz P. (2019). Single cell analysis reveals immune cell–adipocyte crosstalk regulating the transcription of thermogenic adipocytes. eLife, 8, e49501.

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Isolation and Characterization of Monocyte-Derived Macrophages

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Frozen peripheral blood mononuclear cells (PBMCs) from healthy donors and patients were thawed and CD14⁺ cells selected using magnetic beads (Miltenyi). 2 x 10⁵ cells/ well in a 24 well plate were seeded on 10ug/ml fibronectin-coated cover slips (R&D systems) in 500ul 20ng/ml macrophage colony stimulating factor (MCSF, Gibco) for 6 days to obtain monocyte-derived macrophages (MDMs). Cells were fixed with paraformaldehyde 4% (Thermo Fisher Scientific) for 10 minutes on ice followed by 8% for 20 minutes at room temperature, permeabilised with 0.1% triton (Sigma) for 5 minutes at room temperature and non-specific binding reduced by blocking with 5% BSA/PBS for 1 hour at room temperature. Cells were incubated with primary anti-vinculin antibody (Sigma 1:200) for 1 hour at room temperature, washed twice with PBS and incubated with secondary antibody conjugated to Alexa Fluor 488 (1:500 Life Technologies) and phalloidin-conjugated to Alexa Fluor 633 (1:200 Thermo Fisher Scientific) for one hour at room temperature. Cells were washed twice with PBS and cover slips mounted onto slides using mounting solution with DAPI for nuclear staining (ProLong Diamond Antifade Mountant with DAPI, Life Technologies) overnight. Slides were imaged using Zeiss 710 confocal microscope at 63x magnification and podosome analysis was carried out on at least 100 cells per sample from 10 fields of view.

Thaventhiran J.E., Allen H.L., Burren O.S., Rae W., Greene D., Staples E., Zhang Z., Farmery J.H., Simeoni I., Rivers E., Maimaris J., Penkett C.J., Stephens J., Deevi S.V., Sanchis-Juan A., Gleadall N.S., Thomas M.J., Sargur R.B., Gordins P., Baxendale H.E., Brown M., Tuijnenburg P., Worth A., Hanson S., Linger R., Buckland M.S., Rayner-Matthews P.J., Gilmour K.C., Samarghitean C., Seneviratne S.L., Sansom D.M., Lynch A.G., Megy K., Ellinghaus E., Ellinghaus D., Jorgensen S.F., Karlsen T.H., Stirrups K.E., Cutler A.J., Kumararatne D.S., Chandra A., Edgar J.D., Herwadkar A., Cooper N., Grigoriadou S., Huissoon A., Goddard S., Jolles S., Schuetz C., Boschann F., Lyons P.A., Hurles M.E., Savic S., Burns S.O., Kuijpers T.W., Turro E., Ouwehand W.H., Thrasher A.J, & Smith K.G. (2020). Whole genome sequencing of a sporadic primary immunodeficiency cohort. Nature, 583(7814), 90-95.

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Immunofluorescence Assay for ZIKV Infection

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Infected cells from the replication growth kinetics assay were fixed with 4% PFA at days 4, 7, 11 and 17 post infection, permeabilized with 70% ethanol and stained using an immunofluorescence assay (IFA) as described previously^{25 (link)}. Briefly, cells were incubated with mouse monoclonal antibody anti-flavivirus group antigen (MAB10216) clone D1–4G2-4-15 (Millipore, Germany) followed by staining with goat anti-mouse IgG conjugated with Alexa Fluor 488 (Life technologies, the Netherlands). After incubation, cells were mounted in ProLong® Diamond Antifade Mountant with DAPI (Life technologies, USA). Uninfected cells and ZIKV-infected cells stained with mouse isotype IgG2a antibody (Dako, Denmark) were used as negative controls. ZIKV-infected cells were identified by using a Zeiss LSM 700 confocal laser scanning microscope fitted on an Axio observer Z1 inverted microscope (Zeiss, Breda, the Netherlands). All images were processed using Zen 2010 software (Zeiss).

Mumtaz N., Koedam M., van den Doel P.B., van Leeuwen J.P., Koopmans M.P., van der Eerden B.C, & Rockx B. (2018). Zika virus infection perturbs osteoblast function. Scientific Reports, 8, 16975.

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Isolation and Characterization of Monocyte-Derived Macrophages

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Visualizing Actin Cytoskeleton Dynamics

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Cells were seeded on coverslips and treated for 48 hours with siRNA as indicated. 6 hours after wounds were made, cells were fixed with 4% paraformaldehyde in PBS for 10 minutes and blocked with 0.1% Triton X-100 and 1% BSA in PBS for 20 minutes. This was followed by incubation with Alexa Fluor® 488 Phalloidin (Life Technologies) in 0.1% Triton X-100 and 1% BSA in PBS for 20 minutes to stain filamentous actin. Finally cells were mounted with ProLong® Diamond Antifade Mountant with DAPI (Life Technologies). The LSM 700 confocal laser scanning microscope (Zeiss) was used for analysis.

Mirabello L., Koster R., Moriarity B.S., Spector L.G., Meltzer P.S., Gary J., Machiela M.J., Pankratz N., Panagiotou O.A., Largaespada D., Wang Z., Gastier-Foster J.M., Gorlick R., Khanna C., de Toledo S.R., Petrilli A.S., Patiño-Garcia A., Sierrasesúmaga L., Lecanda F., Andrulis I.L., Wunder J.S., Gokgoz N., Serra M., Hattinger C., Picci P., Scotlandi K., Flanagan A.M., Tirabosco R., Amary M.F., Halai D., Ballinger M.L., Thomas D.M., Davis S., Barkauskas D.A., Marina N., Helman L., Otto G.M., Becklin K.L., Wolf N.K., Weg M.T., Tucker M., Wacholder S., Fraumeni JF J.r., Caporaso N.E., Boland J.F., Hicks B.D., Vogt A., Burdett L., Yeager M., Hoover R.N., Chanock S.J, & Savage S.A. (2015). A genome-wide scan identifies variants in NFIB associated with metastasis in patients with osteosarcoma. Cancer discovery, 5(9), 920-931.

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Quantifying RNA Foci in DM1 Myotubes

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Myotubes or slices of vastus lateralis (quadriceps) muscle from HSA^LR mice were fixed with 4% w/v PFA, permeabilized using 70% v/v ethanol, and pre-hybridized for 30 min at 37 °C. Myotubes were probed for 4 hrs at 50 °C with a Cy3-(CAG)₈ probe (IDT). Slides were washed with 42 °C pre-warmed 40% v/v formamide in 2X SSC and mounted using ProLong Diamond Antifade mountant with DAPI (Life Technologies). Myotubes were imaged on a Zeiss LSM 840 confocal scanning microscope with a 40X water objective. Number of nuclear foci for each cell was quantified using Fiji. In DM1 myotubes, the foci were counted blind in at least 100 nuclei per replicate (at least 300 nuclei over three replicates) for all treatments. For the muscle from HSA^LR mice, the number of total nuclei was divided by the number of nuclei containing foci to calculate the percent number of nuclei with foci and at least 100 nuclei per replicate (at least 300 nuclei over three replicates) were counted blind for all treatments.

Jenquin J.R., Yang H., Huigens III R.W., Nakamori M, & Berglund J.A. (2019). Combination Treatment of Erythromycin and Furamidine Provides Additive and Synergistic Rescue of Mis-splicing in Myotonic Dystrophy Type 1 Models. ACS Pharmacology & Translational Science, 2(4), 247-263.

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