Image it signal enhancer

Manufactured by Thermo Fisher Scientific

Sourced in France, United Kingdom, United States

The Image-iT Signal Enhancer is a laboratory reagent that is designed to amplify and enhance fluorescent signals in microscopy applications. It is a simple, ready-to-use solution that can be applied directly to fixed and permeabilized cells or tissue samples to improve the visualization and detection of fluorescently labeled targets.

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Lab products found in correlation

Turbofect, by Thermo Fisher Scientific (2 mentions) Lsm 700, by Zeiss (2 mentions) Click it edu reaction additive, by Thermo Fisher Scientific (2 mentions) Axiovert microscope, by Zeiss (2 mentions) Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions) Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (1 mentions) Ssea 1, by Developmental Studies Hybridoma Bank (1 mentions) Hoechst 33342, by Dojindo Laboratories (1 mentions) Eclipse 80i microscope, by Nikon (1 mentions) Glass chamber slide, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 azide, by Thermo Fisher Scientific (1 mentions) Fv100mpe microscope, by Olympus (1 mentions) Alexa fluor555 azide, by Thermo Fisher Scientific (1 mentions) Chondroitinase b, by R&D Systems (1 mentions) Alexa fluor 488 conjugated goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions) F view digital camera, by Olympus (1 mentions) Bx61 microscope, by Olympus (1 mentions) Chondroitinase abc, by Seikagaku (1 mentions) Chondroitinase acii, by Seikagaku (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Image-iTTM FX Signal Enhancer Image-iT

12 protocols using image it signal enhancer

Fluorescent Immunohistochemistry of Embryos

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Primary antibodies used were chicken polyclonal anti-GFP (Abcam, ab13970, 1:500), rabbit anti-GFP (Molecular Probes A6465, 1:500) and rabbit anti-activated Caspase-3 (Fisher Scientific/BD, BDB559565, 1:500). Antibody used for fluorescent in situ hybridization was mouse anti-Dig (Jackson ImmunoResearch 200-002-156, 1:5000), detected with Invitrogen Tyramide #5 (ThermoFisher Scientific, T20915). Secondary antibodies used were Alexa Fluor 568 goat anti-mouse (ThermoFisher Scientific, A-11031, 1:500), Alexa Fluor 488 goat anti-rabbit (ThermoFisher Scientific, A-11034, 1:500) and Alexa Fluor 488 goat anti-chicken IgG (H+L) (ThermoFisher Scientific, A-11039, 1:500).
Embryos for immunohistochemistry were treated with acetone for 20 min to permeabilize them, then washed for 5 min in distilled water and 2 × 10 min in PBS. Embryos were treated with Image-iT Signal Enhancer (ThermoFisher Scientific, I36933) for 30 min, then incubated in block solution (2 % goat serum, 1 % BSA, 10 % DMSO and 0.5 % Triton) for 1 h followed by incubation in primary antibody in fresh block solution at 4°C overnight. Embryos were washed with PBT (PBS + 0.1 % Triton) for 2 h and incubated with secondary antibody in block solution at 4°C overnight. Embryos were then washed with PBT for 2 h and stored in 2 % DABCO (Acros Organics, AC112471000).

Juárez-Morales J.L., Weierud F., England S.J., Demby C., Santos N., Grieb G., Mazan S, & Lewis K.E. (2021). Evolution of lbx spinal cord expression and function. Evolution & development, 23(5), 404-422.

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Imaging Cellular Protein Localization

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NUP samples were prepared as described previously^{23 (link)}. In brief, U2OS cell stably expressing Nup96-SNAP (ATCC-HTB-96, Lot # 61074667) were seeded on coverslips. After a prefixation (2.4% PFA; 30 s) and permeabilization (0.4% Triton X-100; 3 min), cells were fixed in 2.4% PFA for 30 min. After quenching the PFA (50 mM NH₄Cl) and washing (PBS; 2x 5 min), blocking was performed with ImageIT Signal Enhancer (Thermo Fisher) for 30 min. For staining, cells were incubated with 1 µM SNAP-Surface Alexa Fluor 647 (Thermo Fisher) in PBS/0.5% BSA/1 mM DTT for 50 min. Before mounting, the samples were washed three times with PBS for 5 min. For imaging clathrin coated vesicles, HeLa cells were transfected with SNAP-CLC^{33 (link)} using Turbofect (Thermo Fisher) according to the manufacturers' protocol. Then the cells were labeled as described for Nup96-SNAP cells. The coverslips carrying the cells were prepared as described in the sample mounting and imaging buffers section.

Schmidt R., Weihs T., Wurm C.A., Jansen I., Rehman J., Sahl S.J, & Hell S.W. (2021). MINFLUX nanometer-scale 3D imaging and microsecond-range tracking on a common fluorescence microscope. Nature Communications, 12, 1478.

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Immunofluorescence Staining of Transfected COS-7 Cells

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For immunofluorescence staining experiments, COS-7 cells were seeded on coverslips 1 d before transfection. 24 h after transfection, the cells were incubated with warm extraction buffer (0.2% glutaraldehyde, 0.35% Triton X-100 in MRB80) for 2 min before incubation with fixation buffer (0.1% glutaraldehyde, 4% paraformaldehyde and 4% sucrose [w/v]) for 10 min at room temperature (staining for α-tubulin and EB1) followed by permeabilization with 0.5% Triton X-100 for 10 min. Next, samples were quenched with 100 mM NaBH₄ before blocking with Image-iT Signal Enhancer (Thermo Fisher Scientific) for 30 min at room temperature and sequentially incubated with 1 µM Alexa647-SNAP-dye (NEB) and 1 mM DTT in PBS for 1 h at room temperature. Next, samples were blocked with 1% bovine serum albumin (BSA) diluted in phosphate-buffered saline (PBS) for 50 min at room temperature and sequentially incubated with CF680-GFP-Nanobody for 1 h at room temperature or overnight at 4°C. Coverslips were incubated with gold nanoparticles (Nanopartz) for 5 min. Then, the coverslips were mounted in GLOX buffer supplemented with 56 mM MEA and sealed with glue (Picodent Twinsil).

van den Berg C.M., Volkov V.A., Schnorrenberg S., Huang Z., Stecker K.E., Grigoriev I., Gilani S., Frikstad K.A., Patzke S., Zimmermann T., Dogterom M, & Akhmanova A. (2023). CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal side. The Journal of Cell Biology, 222(4), e202208062.

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Immunofluorescence of Cellular Proteins

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Cells were cultured for 36 h, fixed with 4% paraformaldehyde, and blocked with Image-iT signal enhancer (Thermo: I36933). Thereafter, cells were incubated with primary antibody (Santa Cruz mouse YAP: sc-101199, 1:200; ABclonal rabbit CHRAC1: A14896, 1:200) overnight at 4 °C and secondary antibody for 1 h. For Ki67 staining, freshly dissected tumor tissue was fixed and embedded in OCT reagent (Sakura) and cut to 4µm sections. Thereafter, sections were treated with citrate buffer for antigen retrieval and the following steps are similar to cell immunofluorescence staining.

Li S., Wang L., Shi J., Chen Y., Xiao A., Huo B., Tian W., Zhang S., Yang G., Gong W, & Zhang H. (2024). Chromatin accessibility complex subunit 1 enhances tumor growth by regulating the oncogenic transcription of YAP in breast and cervical cancer. PeerJ, 12, e16752.

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Piezo2 Receptor Immunocytochemistry

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The neurons were fixed by incubation in PLP fixative (2% paraformaldehyde, 75 mM lysine, 37 mM sodium phosphate, 10 mM sodium periodate) for 3 hours at 4°C. The fixed neurons were permeabilized in 0.1% Triton X-100 solution (MP Biomedicals, Santa Ana, CA). The nonspecific bindings of IgG were blocked by incubations in image-iT signal enhancer (Thermo Fisher scientific) and subsequently in a solution containing 5% normal goat serum and 0.2% bovine serum albumin. The neurons were incubated with an anti-Piezo2 rabbit antibody (#sc-84763, Santa Cruz Biotechnology, Dallas, TX) or the anti-Piezo2 antibody which was treated with its antigen. The neurons were further incubated with an Alexa 488-conjugated F(ab')2 fragment of goat anti-rabbit IgG(H+L) (Thermo Fisher) and DAPI. Images were captured with a confocal laser scanning microscope (LSM700, Carl Zeiss, Oberkochen,
Germany).

Yamaguchi S, & Otsuguro K. (2017). A mechanically activated ion channel is functionally expressed in the MrgprB4 positive sensory neurons, which detect stroking of hairy skin in mice. Neuroscience letters, 653.

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Immunofluorescence Staining of Avian PGCs

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Circulating PGCs derived from HH14 embryonic blood or cultured PGCs were adhered onto a MAS-GP Type A coated
glass slide (Matsunami Glass, Osaka, Japan) and fixed with 4% paraformaldehyde for 5 min at room temperature
(RT). After several washes, cells were blocked with PBS containing 5% normal goat serum or Image-iT signal
enhancer (Life Technologies) and incubated overnight at 4ºC with primary antibodies. Then, cells were
incubated for 30 min or 1 h at RT with secondary antibodies. Subsequently, cells were counterstained with 1
µg/ml Hoechst 33342 (Dojindo, Kumamoto, Japan). Fluorescent images were captured using an Eclipse E1000
fluorescence upright microscope (Nikon, Tokyo, Japan), and these images were processed using Photoshop
Elements (Adobe Systems, San Jose, CA, USA) for trimming and overlaying. Sources and dilution of used
antibodies were as follows: rat anti-chicken vasa homolog (CVH) raised in our laboratory (1:10000) [27 (link)], mouse anti-stage specific embryonic antigen-1 (SSEA-1; 1:100,
Developmental Studies Hybridoma Bank, Iowa City, IA, USA), mouse anti-chicken c-KIT (1:500, SouthernBiotech,
Birmingham, AL, USA), goat anti-rat IgG conjugated with Alexa Fluor 488 (1:1000, Life Technologies) and goat
anti-mouse IgG or IgM conjugated with Alexa Fluor 594 (1:1000, Life Technologies).

MIYAHARA D., OISHI I., MAKINO R., KURUMISAWA N., NAKAYA R., ONO T., KAGAMI H, & TAGAMI T. (2015). Chicken stem cell factor enhances primordial germ cell proliferation cooperatively with fibroblast growth factor 2. The Journal of Reproduction and Development, 62(2), 143-149.

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Quantification of Viral Genome Replication

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Primary HMVEC-d and HFF cells seeded on glass chamber slides (Nalgene Nunc International) were uninfected, KSHV infected (30 DNA copies/cell), or HSV-1 infected (1pfu/cell), fixed for 15 min with 4% paraformaldehyde, and permeabilized using 0.2% Triton X-100 for 5 min. Cells were then washed and blocked using Image-iT signal enhancer (Life Technologies) for ~20 min followed by incubation with primary antibodies and then incubated with secondary antibodies conjugated with fluorescent dye. To detect EdU labeled viral genome, cells were fixed, permeabilized and blocked with Image-iT signal enhancer for 20 min. A CLICK reaction was performed for 30 min at RT using Click-iT EdU reaction additive (Life Technologies), copper sulphate, EdU reaction buffer and Alexa Fluor 594 azide. Cells were observed by Nikon Eclipse 80i microscope, and analyzed with Metamorph digital imaging software. All images were acquired at 40X magnification.

Iqbal J., Ansari M.A., Kumar B., Dutta D., Roy A., Chikoti L., Pisano G., Dutta S., Vahedi S., Veettil M.V, & Chandran B. (2016). Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses. PLoS Pathogens, 12(10), e1005967.

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Viral Genome and RNA Detection Assay

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Mouse BMDMs were seeded on glass chamber slides (ThermoFisher Scientific) infected with or without HSV-1 (MOI, 10). Cells were then washed and blocked using Image-iT signal enhancer (Life Technologies) for 20 min, followed by incubation with primary antibody (Invitrogen, PA5-86634), and then incubated with secondary antibodies conjugated with fluorescent dye. To detect EdU labeled viral genome, cells were fixed, permeabilized, and blocked with Image-iT signal enhancer for 20 min. A CLICK reaction was performed for 30 min at RT using Click-iT EdU reaction additive (Life Technologies), copper sulfate, EdU reaction buffer, and Alexa Fluor 555 azide (ThermoFisher Scientific). To detect EU-labeled IAV, a CLICK reaction was performed using Click-iT EU reaction additive (Life Technologies), copper sulfate, EU reaction buffer, and Alexa Fluor 488 azide according to the manufacturer’s instructions (ThermoFisher Scientific). Cells were observed by Olympus FV100MPE microscope, and analyzed with FV10-ASW_Viewer imaging software.

Jiang Y., Sun S., Quan Y., Wang X., You Y., Zhang X., Zhang Y., Liu Y., Wang B., Xu H, & Cao X. (2023). Nuclear RPSA senses viral nucleic acids to promote the innate inflammatory response. Nature Communications, 14, 8455.

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Fluorescent Immunocytochemistry Protocol

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Cells were gently washed in prewarmed D-PBS, fixed in 4% paraformaldehyde for 30 min at 37°C, then permeabilized with 0.5% Triton X-100. Preparations were saturated 30 min with Image-iT Signal Enhancer (InVitrogen Life Technology, France). Primary and secondary antibodies were diluted in background reducingbuffer (Dako antibody reagent with background reducer, InVitrogen Life Technology, France). Immuno-labelled cells were mounted in a ProLong Antifade Gold solution (InVitrogen Life Technology, France) before analyses.

Ponceau A., Albigès-Rizo C., Colin-Aronovicz Y., Destaing O, & Lecomte M.C. (2015). αII-Spectrin Regulates Invadosome Stability and Extracellular Matrix Degradation. PLoS ONE, 10(4), e0120781.

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Corneal Extracellular Matrix Mapping

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Small, full-thickness samples of bovine cornea were cut from the central 0–3 mm zone, the 3–6 mm zone, the 6–9 mm zone, and the 9–12 mm zone and embedded in OCT. Cryosections, 8 μm thick, were blocked with an Image IT Signal Enhancer (Invitrogen, UK)/5% normal goat serum for 20 min then incubated with the anti-keratan sulphate primary antibodies 5D4 (1:100 final dilution) and 1B4 (1:50 final dilution). Additional sections were also incubated for 1–2 h at 37 °C with antibody, 2B6 (1:20 dilution)—specific for CS/DS GAGs, with or without enzyme pretreatment to expose the CS and DS epitopes: chondroitinase ABC (0.4 U/ml) for CS and DS GAGs, or chondroitinase ACII (0.1 U/ml) (Seikagaku Corp., Tokyo, Japan) for CS GAGs, or chondroitinase B (0.5 U/ml) (R & D Systems, Inc., MN) for DS GAGs. Sections were then labelled with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody, mounted with Prolong Gold containing the nuclear stain DAPI (Invitrogen, UK) and examined on an Olympus BX61 microscope and F-View digital camera.

Ho L.T., Harris A.M., Tanioka H., Yagi N., Kinoshita S., Caterson B., Quantock A.J., Young R.D, & Meek K.M. (2014). A comparison of glycosaminoglycan distributions, keratan sulphate sulphation patterns and collagen fibril architecture from central to peripheral regions of the bovine cornea. Matrix Biology, 38, 59-68.

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