Image it fx signal enhancer, by Thermo Fisher Scientific - Product details

1

Immunofluorescence Staining and Microscopy

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Cells in 8-chamber slides were infected as indicated before fixation for 10 min with 4% paraformaldehyde. They were washed with PBS, permeabilized with 0.2% Triton X-100 for 10 min, and blocked with Image-IT FX signal enhancer (Life Technologies) for 10 min. Cells were incubated with primary antibodies for 1 hour at room temperature in PBS, 1% BSA, washed, and incubated with Alexa Fluor-conjugated secondary antibodies for 1 hour at room temperature in PBS, 1% BSA. They were washed again and mounted onto slides using Slowfade gold mounting reagent with DAPI (Life Technologies). BrdU-labeled virus was detected as described [93] (link). Briefly, cells were fixed for 10 min with 4% paraformaldehyde then treated with 4N HCl for 10 min at room temperature to expose BrdU to antibody staining. Cells were washed with PBS, permeablized with 0.2% Triton X-100 for 10 min, and blocked with Image-IT FX signal enhancer (Life Technologies) for 10 min. Cells were stained with a rabbit-derived antibody to BrdU and Alexa Fluor-conjugated secondary antibodies (Life Technologies) and mounted onto slides as above. Cells were imaged using a Nikon Eclipse 80i fluorescence microscope and Metamorph software (Molecular Devices, Silicon Valley, CA). Figures shown are representatives of two or more experiments each.

Johnson K.E., Bottero V., Flaherty S., Dutta S., Singh V.V, & Chandran B. (2014). IFI16 Restricts HSV-1 Replication by Accumulating on the HSV-1 Genome, Repressing HSV-1 Gene Expression, and Directly or Indirectly Modulating Histone Modifications. PLoS Pathogens, 10(11), e1004503.

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2

KSHV Infection and Genome Detection in HMVEC-d Cells

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HMVEC-d cells were seeded on glass 8-well chamber slides (Nalgene Nunc International, Naperville, IL) and uninfected or KSHV infected (30 DNA copies/cell or latently infected) cells were fixed for 15 min with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100 for 5 min. Cells were then washed and blocked with Image-iT FX signal enhancer (Life Technologies) for 20 min. The cells were reacted with primary antibodies against the specific proteins, followed by fluorescent dye-conjugated secondary antibodies. To detect EdU labeled viral genome, cells were fixed, permeabilized and blocked with Image-iT FX signal enhancer (Life Technologies) for 20 min. A CLICK reaction was performed for 30 min at RT using Click-iT EdU reaction additive (Life Technologies), EdU reaction buffer, copper sulphate and Alexa Fluor 594 azide. Cells were observed by Nikon Eclipse 80i microscope, and analyzed with Metamorph digital imaging software. All experiments were performed three independent times and three different fields with a minimum of 20 cells were analyzed. All images were acquired at 40 X magnification.

Dutta D., Dutta S., Veettil M.V., Roy A., Ansari M.A., Iqbal J., Chikoti L., Kumar B., Johnson K.E, & Chandran B. (2015). BRCA1 Regulates IFI16 Mediated Nuclear Innate Sensing of Herpes Viral DNA and Subsequent Induction of the Innate Inflammasome and Interferon-β Responses. PLoS Pathogens, 11(6), e1005030.

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3

Immunofluorescence Staining of iPSCs

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For immunofluorescence staining, patient iPSCs were fixed in 4% paraformaldehyde for 15 min, rinsed with DPBS, and permeabilized with 0.3% Triton X-100 in DPBS for 15 min. The cells were then incubated with Image-iT™ FX signal enhancer (Thermo Fisher Scientific) for 40 min at room temperature in a humidified environment and then followed by incubation individually with primary antibodies, including SOX2, OCT4, NANOG and SSEA4, diluted in the Image-iT™ FX signal enhancer blocking buffer, overnight at 4 °C. Cells were then washed and incubated with corresponding secondary antibody conjugated with Alexa Fluor 488 or Alex Fluor 594 for 1 h at room temperature (antibodies used are listed in Table 2). Cells were washed and stained with Hoechst 33342 nucleic acid stain for 15 min and imaged using an INCell Analyzer 2200 imaging system (GE Healthcare) with 20 × objective lens and Texas Red, FITC and DAPI filter sets.

Li R., Pradhan M., Xu M., Roeder A., Beers J., Zou J., Liu C., Porter F.D, & Zheng W. (2020). An induced pluripotent stem cell line (TRNDi001-D) from a Niemann-Pick disease type C1 (NPC1) patient carrying a homozygous p. I1061T (c. 3182T>C) mutation in the NPC1 gene. Stem cell research, 44, 101737.

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Immunofluorescence Staining of Patient iPSCs

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For immunofluorescence staining, patient iPSCs were fixed in 4% paraformaldehyde for 15 min, rinsed with Dulbecco’s phosphate-buffered saline (DPBS), and permeabilized with 0.3% Triton X-100 in DPBS for 15 min. The cells were then incubated with the Image-iT™ FX signal enhancer (ThermoFisher Scientific) for 40 min at room temperature in a humidified environment, followed by incubation with primary antibodies including SOX2, OCT4, NANOG and SSEA4, diluted in the Image-iT™ FX signal enhancer blocking buffer, overnight at 4 °C. After washing with DPBS, a corresponding secondary antibody conjugated with Alexa Fluor 488 or Alex Fluor 594 was added to the cells and incubated for 1 h at room temperature (Antibodies used are listed in Table 2). Cells were washed and then stained with Hoechst 33342 for 15 min and imaged using an INCell Analyzer 2200 imaging system (GE Healthcare) with 20X objective lens and Texas Red, FITC and DAPI filter sets.

Li R., Baskfield A., Lin Y., Beers J., Zou J., Liu C., Jaffré F., Roberts A.E., Ottinger E.A., Kontaridis M.I, & Zheng W. (2018). Generation of an induced pluripotent stem cell line (TRNDi003-A) from a Noonan syndrome with multiple lentigines (NSML) patient carrying a p.Q510P mutation in the PTPN11 gene. Stem cell research, 34, 101374.

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5

Immunofluorescence Staining of iPSCs

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For immunofluorescence staining, patient iPSCs were fixed in 4% paraformaldehyde for 15 min, rinsed with DPBS, and permeabilized with 0.3% Triton X-100 in DPBS for 15 min. The cells were then incubated with Image-iT™ FX signal enhancer (Thermo Fisher Scientific) for 40 min at room temperature in a humidified environment and then followed by incubation individually with primary antibodies, including SOX2, OCT4, NANOG and SSEA4, diluted in the Image-iT™ FX signal enhancer blocking buffer, overnight at 4 °C. Cells were then washed and incubated with corresponding secondary antibody conjugated with Alexa Fluor 488 or Alex Fluor 594 for 1 h at room temperature (antibodies used are listed in Table 2). Cells were washed and stained with Hoechst 33342 nucleic acid stain for 15 min and imaged using an INCell Analyzer 2200 imaging system (GE Healthcare) with 20 × objective lens and Texas Red, FITC and DAPI filter sets.

Li R., Pradhan M., Xu M., Roeder A., Beers J., Zou J., Liu C., Porter F.D, & Zheng W. (2020). An induced pluripotent stem cell line (TRNDi001-D) from a Niemann-Pick disease type C1 (NPC1) patient carrying a homozygous p. I1061T (c. 3182T>C) mutation in the NPC1 gene. Stem cell research, 44, 101737.

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iPSC Pluripotency Marker Immunostaining

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For immunofluorescence staining, patient iPSCs were fixed in 4% paraformaldehyde for 15 mins, rinsed with Dulbecco’s Phosphate Buffered Saline (DPBS), and permeabilized with 0.3% Triton X-100 in DPBS for 15 mins. The cells were then incubated with the Image-iT™ FX signal enhancer (Thermo Fisher Scientific) for 40 mins at room temperature in a humidified environment and then followed by incubation individually with primary antibodies including SOX2, OCT4, NANOG and SSEA4, diluted in the Image-iT™ FX signal enhancer blocking buffer, overnight at 4 °C. After washing with DPBS, a corresponding secondary antibody conjugated with Alexa Fluor 488 or Alex Fluor 594 was added to the cells and incubated for 1 h at room temperature (Antibodies used are listed in Table 2). Cells were then washed and stained with Hoechst 33342 nucleic acid stain for 15 mins and imaged using an INCell Analyzer 2200 imaging system (GE Healthcare) with 20× objective lens and Texas Red, FITC and DAPI filter sets.

Li R., Baskfield A., Beers J., Zou J., Liu C., Alméciga-Díaz C.J, & Zheng W. (2019). Generation of an induced pluripotent stem cell line (TRNDi005-A) from a Mucopolysaccharidosis Type IVA (MPS IVA) patient carrying compound heterozygous p.R61W and p.WT405del mutations in the GALNS gene. Stem cell research, 36, 101408.

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iPSC Pluripotency Marker Immunostaining

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For immunofluorescence staining, patient iPSCs were fixed in 4% paraformaldehyde for 15 mins, rinsed with Dulbecco’s Phosphate Buffered Saline (DPBS), and permeabilized with 0.3% Triton X-100 in DPBS for 15 mins. The cells were then incubated with the Image-iT™ FX signal enhancer (Thermo Fisher Scientific) for 40 mins at room temperature in a humidified environment and then followed by incubation individually with primary antibodies including SOX2, OCT4, NANOG and SSEA4, diluted in the Image-iT™ FX signal enhancer blocking buffer, overnight at 4 °C. After washing with DPBS, a corresponding secondary antibody conjugated with Alexa Fluor 488 or Alex Fluor 594 was added to the cells and incubated for 1 h at room temperature (Antibodies used are listed in Table 2). Cells were then washed and stained with Hoechst 33342 nucleic acid stain for 15 mins and imaged using an INCell Analyzer 2200 imaging system (GE Healthcare) with 20× objective lens and Texas Red, FITC and DAPI filter sets.

Li R., Baskfield A., Beers J., Zou J., Liu C., Alméciga-Díaz C.J, & Zheng W. (2019). Generation of an induced pluripotent stem cell line (TRNDi005-A) from a Mucopolysaccharidosis Type IVA (MPS IVA) patient carrying compound heterozygous p.R61W and p.WT405del mutations in the GALNS gene. Stem cell research, 36, 101408.

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Immunofluorescence and Immunohistochemistry of Liver Sections

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IFM was performed with a Zeiss LSM 510 confocal microscope with a 63x oil objective as previously described.^{[18 ]} Briefly, unstained liver sections were deparaffinized and rehydrated, boiled in antigen unmasking solution (Vector Laboratories, Burlingame, CA), quenched with Image-iT FX signal enhancer (Life Technologies, Grand Island, NY), and blocked for 1 hour. Slides were then incubated overnight at 4°C with cytokeratin (CK) 19 (goat polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA), CK7 (mouse monoclonal IgG₁, AbCam, Cambridge, MA), alpha-smooth muscle actin (rabbit monoclonal, AbCam), and/or IL-10 and IL-10 receptor (goat polyclonal, Sigma-Aldrich, St. Louis, MO) primary antibodies followed by incubation with corresponding fluorophore-conjugated secondary antibodies (Life Technologies). Coverslips were mounted using ProLong Gold antifade reagent (Life Technologies) with DAPI. For IHC, paraffin embedded tissues were deparaffinized and rehydrated, and boiled in antigen unmasking solution as above. Slides were then incubated overnight at 4°C with CD4 (rabbit polyclonal, Novus Biologicals), CD8 (rabbit monoclonal, Abgent) or anti-neutrophil (rat monoclonal, Cedarlane) primary antibodies followed by incubation with HRP-tagged secondary antibodies and developed using the ImmPACT NovaRed substrate kit (Vector Laboratories).

Tabibian J.H., O’Hara S.P., Trussoni C.E., Tietz P.S., Splinter P.L., Mounajjed T., Hagey L.R, & LaRusso N.F. (2015). Absence of the intestinal microbiota exacerbates hepatobiliary disease in a murine model of primary sclerosing cholangitis. Hepatology (Baltimore, Md.), 63(1), 185-196.

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Immunofluorescence Staining of Formalin-Fixed Tissues

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For the staining of tissues, mice were sacrificed and transcardially perfused with saline, followed by 10% neutral buffered formalin (pH 7.4). Formalin-fixed tissues were then embedded in paraffin and sectioned. For immunofluorescence staining, paraffin-embedded sections were deparaffinized, and blocking was performed in Image-iT FX Signal Enhancer (Life Technologies) for 30 min. Primary antibodies were obtained from Abcam (active caspase-3, doublecortin and Choline Acetyltransferase). Secondary antibodies were obtained from Thermo (Alexa Fluor 488 Goat Anti-rabbit IgG (H + L)). The antibodies were diluted with Can Get Signal Immunostain Solution A (TOYOBO). After the samples had been mounted on a slide with a cover glass, they were observed under a Keyence BZ-9000 fluorescence microscope.

Oka N., Shimada K., Ishii A., Kobayashi N, & Kondo K. (2023). SARS-CoV-2 S1 protein causes brain inflammation by reducing intracerebral acetylcholine production. iScience, 26(6), 106954.

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Extracellular Matrix Immunostaining in Cryosections

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Immunostaining for ECM deposition in cryosectioned samples was performed using primary monoclonal antibodies for collagen II, aggrecan, collagen X, and collagen I (Abcam, Cambridge, UK; collagen X from Sigma–Aldrich). Antigen retrieval was performed for all sections by incubating in 20 µg/mL proteinase K (Sigma–Aldrich) for 10 min at 37°C. Samples for aggrecan and collagen X immunostaining were deglycosylated with 0.75 U/mL chondroitinase ABC (Sigma– Aldrich) for 1.5 h at 37°C. Samples were blocked with Image-iT FX signal enhancer (Life Technologies) and incubated with the primary antibodies overnight at 4°C (1:20 dilution for all primary antibodies). Secondary antibody binding with Alexa Fluor 488-conjugated goat polyclonal anti-mouse IgG (Molecular Probes, Carlsbad, CA) at room temperature for 1 h (1:200 dilution). Lastly, samples were stained with Hoechst (Sigma–Aldrich) for 5 min at room temperature to visualize cell nuclei (n = 12).

Lei J., Trevino E, & Temenoff J. (2016). Cell number and chondrogenesis in human mesenchymal stem cell aggregates is affected by the sulfation level of heparin used as a cell coating. Journal of biomedical materials research. Part A, 104(7), 1817-1829.

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Image it fx signal enhancer

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193 protocols using image it fx signal enhancer

Immunofluorescence Staining and Microscopy

KSHV Infection and Genome Detection in HMVEC-d Cells

Immunofluorescence Staining of iPSCs

Immunofluorescence Staining of Patient iPSCs

Immunofluorescence Staining of iPSCs

iPSC Pluripotency Marker Immunostaining

iPSC Pluripotency Marker Immunostaining

Immunofluorescence and Immunohistochemistry of Liver Sections

Immunofluorescence Staining of Formalin-Fixed Tissues

Extracellular Matrix Immunostaining in Cryosections

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