Alexa fluor 647 anti rabbit igg

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 647 anti-rabbit IgG is a fluorescently labeled secondary antibody. It is designed to bind to rabbit primary antibodies, allowing for detection and visualization in various immunoassays and imaging applications.

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Lab products found in correlation

Alexa fluor 647 anti mouse igg, by Thermo Fisher Scientific (5 mentions) Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (4 mentions) Alexa fluor 568 anti goat igg, by Thermo Fisher Scientific (2 mentions) Alexa fluor 647 anti rat igg, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (2 mentions) Alexa fluor 594 anti rabbit igg, by Thermo Fisher Scientific (2 mentions) Anti gfap z0334, by Agilent Technologies (1 mentions) Fluorescence mounting medium, by Agilent Technologies (1 mentions) 710 confocal microscope, by Zeiss (1 mentions) Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions) Facscanto 2 system, by BD (1 mentions) Anti fc receptor, by BioLegend (1 mentions) Goat serum, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Alexa fluor 488 anti mouse igg, by Abcam (1 mentions) Vimentin, by Abcam (1 mentions) Alexa fluor 647 anti rabbit igg, by Abcam (1 mentions) Ve cadherin, by Merck Group (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Tcs sp8 confocal laser scanning microscope, by Leica (1 mentions)

Spelling variants of this product

Alexa Fluor 647 (1520 citations) Alexa 647 (424 citations) Alexa Fluor 647 donkey anti-rabbit IgG (71 citations) Anti-rabbit Alexa Fluor 647 (38 citations) Alexa Fluors (21 citations) Alexa Fluor 647 IgG (9 citations) Alexa Fluor anti-rabbit (4 citations) IgG Rabbit (4 citations) Rabbit Alexa Fluor 647 (3 citations) Anti-IgG Alexa Fluor 647 (2 citations)

27 protocols using alexa fluor 647 anti rabbit igg

Multimodal Analysis of Brain Tissue Sections

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Thick (100-200um) brain sections were prepared with vibratome and stored
in tissue freezing media (25% Glycerol, 30% Ethylene glycol, 1.38g/L
NaH₂PO₄, 5.48g/L Na₂HPO₄). The
primary antibodies used in this study as follows; anti-RFP/ tdTomato (MBS448092;
MyBioSource), anti-GFAP (Z0334; DAKO), anti-NeuN (MAB377, MilliporeSigma),
anti-Olig2 (AB9610, MilliporeSigma), anti-Sox2 (14-9811-80; eBioscience). The
secondary antibodies were as follows (all from Invitrogen): Alexa Fluor 568
anti-goat IgG, Alexa Fluor 647 anti-rat IgG, Alexa Fluor 647 anti-rabbit IgG,
Alexa Fluor 647 anti-mouse IgG. The nucleus was stained with DAPI.

Hara T, & Verma I.M. (2019). Modeling Gliomas Using Two Recombinases. Cancer research, 79(15), 3983-3991.

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Immunofluorescence Staining of α-Synuclein and TH

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Immunofluorescence staining for α-Syn and tyrosine hydroxylase (TH), a marker for dopaminergic neurons, was performed by washing free-floating sections with PBS-T (0.1% Tween 20 or 0.2% Triton X-100) three times for 10 min each at room temperature. Sections were then incubated in blocking solution (1% or 2% BSA, 10% normal goat serum in PBS-T) for 1 h. After blocking, sections were incubated with primary antibody (anti-α-Syn (1:200; Mouse mAb, 32-810, clone SYN 211, Invitrogen); anti-TH (1:500, Rabbit pAb, AB152, Millipore Sigma)) in antibody solution (2% normal goat serum in PBS-T) overnight at RT. Sections were then washed in PBS or PBS-T and incubated with secondary antibody (AlexaFluor™647 anti-mouse IgG (1:500, A21236, Invitrogen); AlexaFluor™647 anti-rabbit IgG (1:500, A21245, Invitrogen)) diluted in antibody solution for 1 h in the dark at room temperature. Sections were then mounted onto glass slides and allowed to dry and then fluorescence mounting medium (DAKO) was applied, followed by cover slips.

Lee E.J., Aguirre-Padilla D.H., Fomenko A., Pawar G., Kapadia M., George J., Lozano A.M., Hamani C., Kalia L.V, & Kalia S.K. (2024). Reduction of alpha-synuclein oligomers in preclinical models of Parkinson's disease by electrical stimulation in vitro and deep brain stimulation in vivo. Brain stimulation, 17(2).

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Quantifying FSTL1 in Osteoarthritic Cartilage

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In accordance with Institutional Review Board regulations, knee joint tissues from OA patients undergoing total knee replacement (University of Iowa Department of Orthopaedics and Rehabilitation) were deidentified and collected as surgical discards. High Mankin (HM) score (mean = 11.7) vs. low Mankin (LM) score (mean = 2.0) tissue was defined as previously described (21). Frozen cartilage tissue was sectioned at 6 μm in a cryostat with the CryoJane Tape Transfer system (Instrumedics) as previously described (22). FSTL1 was labeled with rabbit anti‐human FSTL1 antibody (Pierce) and visualized with Alexa Fluor 647‐anti‐rabbit IgG (Invitrogen). Nuclei were counterstained with Hoechst 33342 dye (Invitrogen), and the coverslips were mounted onto slides. The slides were imaged using a Zeiss 710 confocal microscope with an oil‐immersion objective (63x). Fluorescence intensity was quantified using ImageJ software. The mean fluorescence from at least 30 cells per individual from both LM and HM cartilage was used to calculate the average fluorescence signal.

Chaly Y., Hostager B., Smith S, & Hirsch R. (2020). The Follistatin‐like Protein 1 Pathway Is Important for Maintaining Healthy Articular Cartilage. ACR Open Rheumatology, 2(7), 407-414.

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Brain cell staining and analysis

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For brain cells staining, the Gca‐Cre‐EGFP mice were first injected with 2 ug of phycoerythrin (PE)–conjugated anti‐CD45 antibody as previously described.^[⁵⁴ (link)
^] After 10 min, mice were euthanized. Brains were infused, minced and digested for 30 min with 1.5 mg mL⁻¹ collagenase IV and 0.2 mg mL⁻¹ DNase I at 37 °C. After centrifugation and washing, the pellet was first incubated with an anti‐Fc receptor (Biolegend, San Diego, CA) to reduce nonspecific binding of antibodies, followed by incubation with the indicated antibodies for 20–30 min at 4 °C. Then, cells were stained with PE‐CY7‐CD45 and BV510‐CD11b (Biolegend, San Diego, USA) for 30 min at 4 °C. For CCR10 staining, obtained bone marrow cells were stained with the CCR10 antibody (Proteintech, Rosemont, USA) for 1 h at 4 °C. The cells were further incubated with Alexa fluor 647‐anti rabbit IgG (Invitrogen, Carlsbad, USA) and APC‐Cy7‐CD45, BV510‐CD11b, PE‐Cy7‐Ly6C, PE‐Ly6G (Biolegend, San Diego, USA) for 30 min at 4 °C. All antibodies were diluted according to the manual from the manufacturer’ s website. Stained cells were collected by BD FACSCanto II system and analyzed by FlowJo V10. Dead cells and doublets were removed by FSC‐A/FSC‐H gating. Data were analyzed by FlowJo V10 (BD Biosciences, San Jose, USA).

Zhou R., Wang L., Chen L., Feng X., Zhou R., Xiang P., Wen J., Huang Y, & Zhou H. (2023). Bone Marrow‐Derived GCA+ Immune Cells Drive Alzheimer's Disease Progression. Advanced Science, 10(36), 2303402.

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Quantifying α-SMA Expression in HPaStec Cells

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HPaStec cells (1 × 10⁴) were seeded in 8-well chamber slides (Lab Tek II system, Thermo Fisher Scientific), and the next day were left untreated or were treated with HPSC or HPaStec sEVs (20 μg/ml) for 72 h. Cells were fixed with 100% prechilled methanol, permeabilized with 0.2% Triton X-100, blocked with 10% goat serum (Invitrogen) in PBS, and incubated overnight at 4 °C with the anti-SMA antibody (Thermo Scientific,). Cells were incubated with secondary antibody (Alexa Fluor 647 anti-rabbit IgG, Invitrogen) and were counterstained and mounted with antifade-containing DAPI (Invitrogen). Micrograph images were acquired as described above in the sEVs uptake section. Three independent experiments have been done and the immunofluorescence signal of α-SMA in control and treated wells have been quantified using image J software. Western blot: For an additional validation of αSMA expressions, HPaStec cells were left untreated or were treated with HPSC or HPaStec sEVs (20 μg/ml) for 72 h. After 72 h, cells were harvested, lysed, and Western blot was carried out to determine α-SMA expression as described above in the Western blot section.

Sarcar B., Fang B., Izumi V., O. Nunez Lopez Y., Tassielli A., Pratley R., Jeong D., Permuth J.B., Koomen J.M., Fleming J.B, & Stewart P.A. (2022). A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles. Molecular & Cellular Proteomics : MCP, 21(12), 100438.

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Immunofluorescence Staining of Cell Lines

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HSC-4 cells cultured on cover glass (Matsunami, #5001) were washed with PBS, fixed with ice-cold acetone/methanol (1:1), blocked with 2% FBS for 40 min, and incubated with primary antibodies to E-cadherin (Cell Signaling Technology, #14472S) and vimentin (Abcam, #ab2547). The molecules were then visualized with secondary antibodies: Alexa Fluor-488 anti-mouse IgG (Invitrogen, #A21202) and Alexa Fluor-647 anti-rabbit IgG (Invitrogen, #A31573). Nuclei were stained with Hoechst 33342 (Invitrogen, #H1399). HUAEC monolayers cultured on collagen type I-coated cover glasses (IWAKI, #11-0071) were washed with PBS, fixed with 4% paraformaldehyde solution for 15 min, and permeabilized with 0.1% Triton X-100 for 15 min. The samples were then blocked with 2% FBS for 40 min and incubated with anti-VE-cadherin (Sigma-Aldrich, #MABT129) and SM22α (Abcam, #GR3321006-1) primary antibodies, followed by incubation with secondary antibodies: Alexa Fluor-488 anti-mouse IgG and Alexa Fluor-647 anti-rabbit IgG. Hoechst 33342 was used to visualize nuclei. Stained HSC-4 cells or HUAECs were mounted and observed with a Keyence BZ-X710 fluorescence microscope or a Leica TCS SP8 confocal laser scanning microscope. Quantitative analysis of obtained images (five fields of view from at least four independent samples) was done with Fiji (ImageJ).

Kobayashi M., Fujiwara K., Takahashi K., Yoshioka Y., Ochiya T., Podyma-Inoue K.A, & Watabe T. (2022). Transforming growth factor-β-induced secretion of extracellular vesicles from oral cancer cells evokes endothelial barrier instability via endothelial-mesenchymal transition. Inflammation and Regeneration, 42, 38.

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Immunostaining Protocol for Cytoskeletal Proteins

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The following primary antibodies were used: rabbit anti-CAMSAP3 (Tanaka et al., 2012 (link), 1: 100), mouse anti-α-tubulin (clone DM1A, Sigma-Aldrich T9026, 1:1000), rabbit monoclonal anti-β-tubulin (clone 9F3, Cell Signaling Technology Cat#2128, 1: 100), mouse anti-γ-tubulin (Sigma-Aldrich T6557, 1:1000), rabbit anti-γ-tubulin (Sigma-Aldrich T3559, 1:500), mouse anti-EZRIN (Abcam 4069, 1:100), rat anti-Odf2 (a gift from Sachiko Tsukita, Osaka University, 1: 2), and mouse monoclonal anti-Chibby (Santa Cruz sc-101551, 1:100). The following secondary antibodies were used: donkey CF-488A anti-rat IgG (Biotium 20027, 1:000) and CF-568 anti-rabbit IgG (Biotium 20698, 1:1000) antibodies; goat Alexa Fluor-488 anti-rat IgG (Invitrogen, A-11006, 1:500), Alexa Fluor-488 anti-rabbit IgG (Invitrogen, A-11034, 1:500), Alexa Fluor-568 anti-mouse IgG (Invitrogen, A-11031, 1:500), Alexa Fluor-594 anti-rabbit IgG (Invitrogen A-11037, 1:500), Alexa Fluor-647 anti-rabbit IgG (Invitrogen, A-21245, 1:500), and Alexa Fluor-647 anti-mouse IgG (Invitrogen, A-21236, 1:1000) antibodies; and goat STAR580 anti-mouse IgG (Abberior 2-0002-005-1, 1:500) and STAR635P anti-rat IgG (Abberior 2-0132-007-5, 1:500). The binding specificity of the anti-CAMSAP3 antibody in the use for IF staining was confirmed by previous studies (Tanaka et al., 2012 (link); Mitsuhata et al., 2021 (link)).

Saito H., Matsukawa-Usami F., Fujimori T., Kimura T., Ide T., Yamamoto T., Shibata T., Onoue K., Okayama S., Yonemura S., Misaki K., Soba Y., Kakui Y., Sato M., Toya M, & Takeichi M. (2021). Tracheal motile cilia in mice require CAMSAP3 for the formation of central microtubule pair and coordinated beating. Molecular Biology of the Cell, 32(20), ar12.

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Lung Tissue Immunofluorescence Staining

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To stain the lung tissues, the lungs were inflated with Tissue-Tek optimal cutting temperature (OCT) compound (Sakura, Tokyo, Japan) diluted 1:4 in PBS, subsequently embedded in OCT compound, and frozen at −80°C. Frozen sections (12 μm) of the isolated lung specimens were prepared, fixed, and permeabilized with the BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences) in accordance with the manufacturer's instructions. The lung specimens were incubated overnight at 4°C with purified anti-mouse IL-33 antibodies (clone Poly5165; BioLegend) and anti-pro surfactant protein C (proSP-C) antibodies (EMD Millipore; Merck KGaA, Darmstadt, Germany). After that, we stained them with Alexa Fluor®568 anti-goat IgG and Alexa Fluor®647 anti-rabbit IgG (Invitrogen, San Diego, CA, USA) for 1 h and co-stained them with DAPI (Sigma Aldrich, St. Louis, MO, USA) for 20 min. Images were analyzed with a FluoView (FV10i) confocal microscope (Olympus, Tokyo, Japan).

Kobari S., Kusakabe T., Momota M., Shibahara T., Hayashi T., Ozasa K., Morita H., Matsumoto K., Saito H., Ito S., Kuroda E, & Ishii K.J. (2020). IL-33 Is Essential for Adjuvant Effect of Hydroxypropyl-β-Cyclodexrin on the Protective Intranasal Influenza Vaccination. Frontiers in Immunology, 11, 360.

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Quantifying Intracellular Glutathione in SH-SY5Y Cells

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We used the chloromethyl reagent 7-amino-4-chloro-methylcoumarine (CMAC) (Life Technologies), which produces a highly fluorescent adduct upon reaction with thiol groups for the evaluation of intracellular GSH in SH-SY5Y cells. Cells were incubated at 37 °C for 15 min with 5 μM CMAC and then incubated with serum-free media for 30 min. The cells were fixed with 4% paraformaldehyde (PFA) and then permeabilized with 0.05% Triton-X100 in the case of multiple staining. Non-specific staining was blocked with the reagent PBS containing 5% BSA/0.1% Tween20, and the cells were then incubated with anti-GTRAP3-18 (Novus Biologicals; NB100-1105) and anti-NOVA1 (Abcam; ab183024) at 1:1000 dilution overnight at 4 °C. After a wash with PBS-Tween20, the cells were labeled with fluorescent-labeled secondary antibodies, Alexa-Fluor 488 anti-goat IgG (Molecular Probes; A11055), and Alexa-Fluor 647 anti-rabbit IgG (Molecular Probes; A31573) at 1:1000 dilutions. Finally, the cells were mounted using Fluoromount-Plus mounting solution (Diagnostic Biosystems) and captured with a Nikon A1 confocal microscope. The fluorescence intensity was analyzed by NIS-Elements Imaging Software (NIS Elements C (Ver. 4.30); Nikon) and an average intensity value of the cells in the field was calculated for further analysis.

Kinoshita C., Kikuchi-Utsumi K., Aoyama K., Suzuki R., Okamoto Y., Matsumura N., Omata D., Maruyama K, & Nakaki T. (2021). Inhibition of miR-96-5p in the mouse brain increases glutathione levels by altering NOVA1 expression. Communications Biology, 4, 182.

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Quantifying Collagen Content in Arterial Walls

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Type I and III collagen content was assessed in MA fixed in PFA 4% by immunofluorescence. Briefly, arteries were incubated with anti-type I or type III collagen antibodies (1:200, Abcam) for 60 min at RT. Segments were washed and incubated with Alexa Fluor 647® anti-rabbit IgG (1:200 dilution, 1h, RT, Molecular Probes). Finally, nuclei were stained with DAPI. After rinsing, MA segments were mounted as described above and visualized with a confocal microscope (SP5 Leica Microsystems) by using x40 objective zoom 2. A minimum of three regions, were randomly selected. To avoid biased selection, the regions were chosen in the DAPI wavelength, as described above. Once selected the images were acquired at identical conditions of brightness, contrast, and laser power at 633 nm excitation/-640-650 nm emission wavelength (secondary antibody-Alexa 647) to detect collagen, either type I or type III. Quantitative analysis of collagen content in vascular wall was performed with Metamorph images analysis software as follows. An extended focus image was reconstructed from the serial images. Thereafter, total and background fluorescence intensity values were measured in the reconstructed image.
Collagen content was then estimated by subtracting the background from the total intensity fluorescence values.

Gil-Ortega M., Martín-Ramos M., Arribas S.M., González M.C., Aránguez I., Ruiz-Gayo M., Somoza B, & Fernández-Alfonso M.S. (2016). Arterial stiffness is associated with adipokine dysregulation in non-hypertensive obese mice. Vascular pharmacology, 77.

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