Alexa fluor 647 donkey anti goat

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 647 donkey anti-goat is a fluorescently labeled secondary antibody used in immunodetection applications. It is designed to specifically bind to goat primary antibodies, allowing for the visualization and detection of target proteins or molecules in a sample.

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Alexa Fluor 647 donkey anti-goat IgG Alexa Fluor® 647 donkey anti-goat IgG (H+L) Donkey anti-goat Alexa 647 Alexa Fluor® 647 goat Donkey anti-Goat 647 Alexa Fluor 647 Alexa 647 Alexa Fluors

31 protocols using alexa fluor 647 donkey anti goat

Immunostaining of Bone Tissue Sections

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Freshly dissected bones were fixed in 4% paraformaldehyde overnight followed by 3-day decalcification in 10% EDTA. Bones were sectioned (5μm for thin sections and 50μm for thick sections) using the CryoJane tape-transfer system (Instrumedics, Ann Arbor, MI). Sections were blocked in PBS with 10% horse serum for 1 hour and then stained overnight with chicken-anti-GFP (Aves, Tigard, OR, 1:1000), anti-CD41-APC (eBioscience, eBioMWReg30, 1:200), goat-anti-c-kit (R&D, 1:400), rabbit-anti-perilipin (Sigma, 1:1000) and/or rabbit-anti-laminin (Abcam, Cambridge, MA, 1:400) antibodies. Donkey-anti-goat Alexa Fluor 647, donkey-anti-chicken Alexa Fluor 488 and/or Donkey-anti-goat Alexa Fluor 647 were used as secondary antibodies (Life Technologies, 1:400). Slides were mounted with anti-fade prolong gold (Life Technologies) and images were acquired with a Zeiss LSM780 confocal microscope.

Zhou B.O., Yu H., Yue R., Zhao Z., Rios J.J., Naveiras O, & Morrison S.J. (2017). Bone marrow adipocytes promote the regeneration of stem cells and hematopoiesis by secreting SCF. Nature cell biology, 19(8), 891-903.

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Immunostaining of Bone Tissue Sections

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Evaluating MINC1 Cytotoxicity in HeLa and A549 Cells

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HeLa and A549 cells were plated on fibronectin-coated coverslips or chamber slides (Nunc, 154534), respectively, at equal densities and treated for 3-48 h with either DMSO or 25 µM MINC1. Next, HeLa cells were fixed with ice-cold methanol and incubated with mouse anti-α-tubulin (Sigma Aldrich, T9026) and goat anti-MgcRacGAP (Abcam, ab2270), using goat anti-mouse Alexa Fluor 488 (Life Technologies, A11017) or donkey anti-goat Alexa Fluor 647 (Life Technologies, A-21447) to detect the primary antibodies, respectively. A549 cells were fixed and stained as described previously [38 (link)], using Alexa-555 Phalloidin (Life Technologies, A34055), Hoechst 33342 (Life Technologies, H1399) and the abovementioned antibodies. Coverslips were mounted with reagent containing DAPI when appropriate (Life Technologies, P36931 and P36930) and imaged using a Nikon 9i fluorescence microscope and NIS elements AR software (Nikon). Color balance was normalized for all images using ImageJ (NIH). The cells were scored for four different morphologies, normal (mononuclear), mitotic (rounded up), multinuclear and dead cells, using at least eight randomly chosen fields per condition.

van Adrichem A.J., Fagerholm A., Turunen L., Lehto A., Saarela J., Koskinen A., Repasky G.A, & Wennerberg K. (2015). Discovery of MINC1, a GTPase-Activating Protein Small Molecule Inhibitor, Targeting MgcRacGAP. Combinatorial Chemistry & High Throughput Screening, 18(1), 3-17.

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Cardiac Cell Immunofluorescence Staining

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Tissues were fixed with 4% paraformaldehyde, permeabilized with 0.2% Tween20, and blocked with 10% FBS. Immunostaining was performed using the following primary antibodies: mouse anti-cardiac Troponin T (cTnT) (ThermoFisher; 1:200), rabbit anti-Connexin 43 (Cx-43) (Abcam; 1:200), mouse anti-α-actinin (Abcam; 1:200), rabbit anti-myosin light chain-2v (Santa Cruz; 1:200), goat anti-caveolin3 (Santa Cruz; 1:100); and the following secondary antibodies: donkey anti-mouse-Alexa Fluor 488 (Abcam; 1:400), donkey anti-rabbit-Alexa Fluor 594 (Life Technologies; 1:200) and donkey anti-goat-Alexa Fluor 647 (Life Technologies; 1:200). Phalloidin-Alexa Fluor 660 (Invitrogen; 1:200) was used to stain F-actin fibers. Conjugated vimentin-Cy3 (Sigma; 1:200) was used to stain for vimentin. Confocal microscopy images were obtained using an Olympus FluoView 1000 laser scanning confocal microscope (Olympus Corporation).
Live and dead staining was performed with CFDA (1:1000, Life Technologies) and Propidium Iodide (75:1000, Life Technologies) in PBS. Viability was calculated as the average intensity of CFDA divided by the sum of average intensities of CFDA and PI. (n≥3)
Sarcomere presence was quantified by average intensity of α-actinin divided by the average intensity of DAPI counterstain. (n=3)

Zhao Y., Rafatian N., Feric N.T., Cox B., Aschar-Sobbi R., Wang E.Y., Aggarwal P., Zhang B., Conant G., Pahnke K.A., Protze S., Lee J.H., Davenport Huyer L., Jekic D., Wickeler A., Naguib H., Keller G.M., Vunjak-Novakovic G., Broeckel U., Backx P.H, & Radisic M. (2019). A platform for generation of chamber specific cardiac tissues and disease modelling. Cell, 176(4), 913-927.e18.

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Immunostaining and Microscopy Analysis of Bone Sections

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Immunostaining was conducted on 6 or 10 μm frozen cryostat sections of the undecalcified head, according to the method of Kawamoto (Leica, CM3050S)^{28 (link)}. Sections were blocked with 1% BSA/PBS and then incubated with goat anti-mouse MMP-9 polyclonal antibody (AF909, R&D) or rabbit anti-mouse collagen type I polyclonal antibody (AB765P, Merck Millipore) overnight at 4 °C, followed by donkey anti-goat Alexa Fluor 647 or donkey anti-rabbit Alexa Fluor 488 secondary antibodies (Thermo Fisher Scientific), respectively. Osteocalcin was detected on demineralized paraffin sections that had been deparaffinized, treated with 1% H₂O₂ in methanol, blocked with 1% BSA/PBS, and incubated with rabbit anti-osteocalcin polyclonal antibody (ALX-210-333, Enzo). Corresponding nonimmune rabbit IgG served as a control. Horse anti-rabbit IgG-HRP secondary antibody (MP-7401, VECTOR) was detected with a diaminobenzidine (DAB) HRP substrate (VECTOR). Sections were observed under a confocal laser microscope (LSM-710, Zeiss; FV3000, Olympus) or a light microscope (BX53, Olympus).

Edamoto M., Kuroda Y., Yoda M., Kawaai K, & Matsuo K. (2019). Trans-pairing between osteoclasts and osteoblasts shapes the cranial base during development. Scientific Reports, 9, 1956.

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Antibody Labeling for Organelle Visualization

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Affinity-purified rabbit anti-AQP5 IgG antibody targeted toward amino acid residues 249 to 265 of rat AQP5 (AB15858) and normal goat IgG antibody (NI02-100UG) were obtained from MilliporeSigma. Normal rabbit IgG antibodies (2729) were obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti-calnexin IgG (LS-B4403) was obtained from LifeSpan BioSciences (Dallas, TX, USA). Rabbit anti-LC3B IgG (MBS9435173) was obtained from MyBioSource (San Diego, CA, USA). Rabbit anti-LIMP-2 IgG (NB400-129) was obtained from Novus Biologicals (Centennial, CO, USA). Mouse anti-TOMM20 IgG (ab56783) was obtained from Abcam (Waltham, MA, USA). Goat anti-connexin 50 IgG (sc-20746) and normal mouse IgG (sc-2025) antibody were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies (goat anti-rabbit Alexa Fluor 488, goat anti-rabbit Alexa Fluor 647, donkey anti-rabbit Alexa Fluor 488, and donkey anti-goat Alexa Fluor 647) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Secondary antibody used for western blotting (goat anti-rabbit DyLight 680, goat anti-mouse DyLight 800, and donkey anti-goat DyLight 800) was obtained from Thermo Fisher Scientific.

Gletten R.B., Cantrell L.S., Bhattacharya S, & Schey K.L. (2022). Lens Aquaporin-5 Inserts Into Bovine Fiber Cell Plasma Membranes Via Unconventional Protein Secretion. Investigative Ophthalmology & Visual Science, 63(8), 5.

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Immunofluorescence Staining of Vascular Markers

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Antibodies and their commercial sources were as follows: anti-CD31 (Agilent Technologies, Santa Clara, CA; M0823, 1:200 dilution), anti- VE-cadherin (R&D Systems, Minneapolis, MN; AF938, 1:200 dilution), anti-PDGFRβ (Cell Signaling, 3169, 1:200 dilution), anti-αSMA (Abcam, Cambridge, MA; ab124964, 1:200 dilution), anti-ZO1 (Thermo Fisher Scientific; 40–2200, 1:100 dilution), donkey anti-rabbit Alexa-Fluor 568, donkey anti-mouse Alexa-Fluor 488, and donkey anti-goat Alexa-Fluor 647 (1:200 dilution, Thermo Fisher Scientific).

Kaushik G., Gil D.A., Torr E., Berge E.S., Soref C., Uhl P., Fontana G., Antosiewicz-Bourget J., Edington C., Schwartz M.P., Griffith L.G., Thomson J.A., Skala M.C., Daly W.T, & Murphy W.L. (2018). Quantitative Label-Free Imaging of 3D Vascular Networks Self-Assembled in Synthetic Hydrogels. Advanced healthcare materials, 8(2), e1801186.

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Immunofluorescent Analysis of Neuroinflammation in SAH

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Mice underwent transcardiac perfusion on day 3 after SAH and brains were stored and fixed in 4% paraformaldehyde. Frozen brain samples were sectioned into 30 μm coronal sections using microtome. Brain slices were then incubated with a blocking buffer (0.4% Triton X-100 TBS-T with 5% donkey serum) for 2 h at room temperature and then incubated with primary antibodies (rabbit anti-NF-kB, #8242, Cell Signaling, Danvers, MA, USA {1:100}; Goat Anti-Iba-1, ab5076, Abcam, MA, USA {1:500}; Goat Anti-GFAP, A-31553, ThermoFisher, Waltham, MA, USA {1:200}; Rat anti-CD68, MCA1957, BIO-RAD, CA, USA {1:500}), followed by incubation at room temperature with secondary antibodies (Donkey anti-Rabbit Alexa Flour 488, A32790, ThermoFisher {1:1000}; Donkey anti-Goat Alexa Fluor 647, A-21447, ThermoFisher {1:1000}; Donkey anti-Rat Alexa Fluor 488, A-21208, ThermoFisher {1:1000}) in TBS-T for 45 min. After rinsing, the brain sections were stained with DAPI and mounted on the slide. The images were taken using Nikon Confocal microscopy. The fluorescent signaling was analyzed using Image J.

Liu M., Jayaraman K., Mehla J., Diwan D., Nelson J.W., Hussein A.E., Vellimana A.K., Abu-Amer Y., Zipfel G.J, & Athiraman U. (2023). Isoflurane Conditioning Provides Protection against Subarachnoid Hemorrhage Induced Delayed Cerebral Ischemia through NF-kB Inhibition. Biomedicines, 11(4), 1163.

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Comprehensive Immunostaining Panel for Neural Cell Types

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Anti-CD31 (Agilent Technologies, Santa Clara, CA), Anti-VE-cadherin (R&D Systems, Minneapolis, MN), Anti-PDGFRγ (Cell Signaling Technologies, Danvers, MA), Anti-αSMA (Abcam, Cambridge, MA), Anti-IBA1 (Abcam, Cambridge, MA), Anti-CD45 (BD Biosciences, San Jose, CA), Anti-γIII Tubulin (R&D Systems, Minneapolis, MN), Anti-GFAP (Abcam, Cambridge, MA), Anti-Nestin (Abcam, Cambridge, MA), Anti-Notch2NL (Santa Cruz Biotechnology, Dallas, TX), Donkey antirabbit Alexa-Fluor 568 (ThermoFisher Scientific, Madison, WI), Donkey anti-mouse Alexa-Fluor 488 (ThermoFisher Scientific, Madison, WI), Donkey anti-goat Alexa-Fluor 647 (ThermoFisher Scientific, Madison, WI).

Kaushik G., Gupta K., Harms V., Torr E., Evans J., Johnson H.J., Soref C., Acevedo-Acevedo S., Antosiewicz-Bourget J., Mamott D., Uhl P., Johnson B.P., Palecek S.P., Beebe D.J., Thomson J.A., Daly W.T, & Murphy W.L. (2020). Engineered Perineural Vascular Plexus for Modeling Developmental Toxicity. Advanced healthcare materials, 9(16), e2000825.

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Multimodal Imaging of Bone Marrow

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Freshly dissected bones were fixed in 4% paraformaldehyde overnight followed by 3-day decalcification in 10% EDTA. Bones were sectioned using the CryoJane tape-transfer system (Instrumedics, St. Louis, MO). Sections were blocked in PBS with 10% horse serum for 1 hr and then stained overnight with goat-anti-Angpt1 (Santa Cruz, Dallas, TX, 1:200), chicken-anti-GFP (Aves, Tigard, OR, 1:1000), anti-CD41-PE (eBioscience, clone eBioMWReg30, 1:200) and/or goat-anti-Osteopontin (R&D, Minneapolis, MN, 1:400) antibodies. Donkey-anti-goat Alexa Fluor 647, donkey-anti-chicken Alexa Fluor 488, and/or Donkey-anti-goat Alexa Fluor 555 were used as secondary antibodies (Invitrogen, Grand Island, NY, 1:400). Slides were mounted with anti-fade prolong gold (Invitrogen) and images were acquired with a LSM780 confocal microscope (Zeiss, San Diego, CA). For thick sections, the specimens were cleared overnight with Benzyl Alcohol/Benzyl Benzoate (1:2) solution (Sigma). 3D reconstruction of bone marrow was achieved by Z stack of tiled images of femoral bone marrow with a Zeiss LSM780 confocal microscope.

Zhou B.O., Ding L, & Morrison S.J. (2015). Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting Angiopoietin-1. eLife, 4, e05521.

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