Alexa fluor 635

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor® 635 is a fluorescent dye produced by Thermo Fisher Scientific. It has an excitation maximum at 633 nm and an emission maximum at 647 nm. Alexa Fluor® 635 is commonly used in various biotechnology applications that require a far-red fluorescent label.

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Alexa Fluors (21 citations) Alexa Fluor 635 phalloidin (20 citations) Alexa Fluor 635 streptavidin (10 citations) Alexa Fluor 635 Goat anti-rabbit (2 citations) Alexa Fluor 635-labeled phalloidin (2 citations) Alexa Fluor-488 or −635 (2 citations)

6 protocols using alexa fluor 635

Amyloid-beta Induced Oxidative Stress

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Aβ peptide (Aβ_1-42) was from Bachem Inc. (Torrance, CA, USA). Fluo4-AM, MitoSOX^TM Red Mitochondrial Superoxide Indicator, MitoTracker^® Orange CMTMRos, anti-rabbit Alexa Fluor^® 488 and anti-mouse Alexa Fluor^® 635 were from Molecular Probes, Inc. (Eugene, OR, USA). Hexafluoro-2-propanol (HFIP) and CMC were from Merck (Darmstadt, Germany), Neurobasal and Dulbecco’s modified essential medium (DMEM), B27 supplement and lipofectamine 2000 were from Gibco (Carlsbad, CA, USA). DOTAP Liposomal Transfection Reagent was from Sigma–Aldrich (Oakville, ON, Canada). Phosphodiester oligonucleotides (ODNs) were from Integrated DNA Technologies (Coralville, IA, USA). The mito-Pericam plasmid was donated by Dr. V. Eisner. Bicinchoninic acid assay (BCA) kit and mHsp-70 antibody were from Pierce Biotechnology (Rockford, IL, USA). Ryanodine was from Alexis (Lausen, Switzerland). PDVF membranes were from Millipore (Bedford, MA, USA). RyR2 antibody and Rhod2-AM was from Thermo-Fisher (Waltham, MA, USA). Gp91 ds-tat was from AnaSpec (Fremont, CA, USA).

SanMartín C.D., Veloso P., Adasme T., Lobos P., Bruna B., Galaz J., García A., Hartel S., Hidalgo C, & Paula-Lima A.C. (2017). RyR2-Mediated Ca2+ Release and Mitochondrial ROS Generation Partake in the Synaptic Dysfunction Caused by Amyloid β Peptide Oligomers. Frontiers in Molecular Neuroscience, 10, 115.

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Autophagy Regulation in Drosophila Cells

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Embryonic primary cell cultures were isolated from gastrulating Dmef2-Gal4, UAS-GFP-Atg8a embryos as described previously [18 (link),23 (link),46 (link),71 (link)], and seeded in 384-well plates at 4 X 10⁴ cells (10ml volume) per well. Each well contained 5ul dsRNA in water (0.25ug dsRNA) targeting a gene from the autophagy network or control dsRNAs targeting lacZ (negative control), Atg18 (positive control) or thread (RNAi knockdown control). Following 20 hours incubation in serum-free M3 medium at 18°C, 30μl of serum-containing medium was added to each well for a final fetal calf serum concentration of 10%. Cells were then cultured for an additional 3 days at 25°C before addition of rapamycin (200 nM) and chloroquine (200uM). Cells were incubated for 12 hrs at 25°C before fixation for 2 hr in 2% formaldehyde. Cells were washed and stained overnight at 4°C with phalloidin Alexa Fluor 635 (Molecular Probes; 1:2000), and DAPI (Sigma, 1:5000), then washed again. All washes were performed in PBT, except for a final rinse in PBS prior to image analysis. This experiment was repeated to give 4 biological replicates of each amplicon.

Zirin J., Nieuwenhuis J., Samsonova A., Tao R, & Perrimon N. (2015). Regulators of Autophagosome Formation in Drosophila Muscles. PLoS Genetics, 11(2), e1005006.

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GLUT4 Translocation Monitoring in Muscle Fibers

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Fibers electroporated with GLUT4myc-eGFP chimera plasmids were seeded on glass coverslips, which was followed by serum deprivation for 3 h. Cells were pre-incubated overnight with 10 μM MCC950 (Calbiochem, Sigma-Aldrich, Burlington, MA, USA), 100 nM insulin (Actrapid, Novo-Nordisk, Denmark), or both. Labeling of surface GLUT4myc in nonpermeabilized cells was performed as described [19 (link),20 (link)]. Briefly, after washing with PBS, the fibers were fixed by incubation for 10 min at room temperature with PBS containing 4% paraformaldehyde (Electron Microscopy Science, Hatfield, PA, USA). Next, the skeletal muscle fibers were rinsed with PBS and blocked for 1 h with PBS-1% BSA at room temperature. GLUT4myc-eGFP levels at the cell surface were detected with the monoclonal anti-myc antibody (1/100 dilution, SC-40; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by treatment with a secondary antibody conjugated with AlexaFluor 635 (1/500 dilution, Molecular Probes, Invitrogen, Carlsbad, CA, USA). Images were obtained using a C2+ confocal microscope system (Nikon Instruments Inc., Minato, Tokyo, Japan). GLUT4myc-eGFP translocation analysis was performed as described in [66 (link)].

Américo-Da-Silva L., Aguilera J., Quinteros-Waltemath O., Sánchez-Aguilera P., Russell J., Cadagan C., Meneses-Valdés R., Sánchez G., Estrada M., Jorquera G., Barrientos G, & Llanos P. (2021). Activation of the NLRP3 Inflammasome Increases the IL-1β Level and Decreases GLUT4 Translocation in Skeletal Muscle during Insulin Resistance. International Journal of Molecular Sciences, 22(19), 10212.

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Immunofluorescence Staining of Mouse Incisor Cryosections

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Immunofluorescence staining used standard protocol on 12 µm sagittal cryosections of mouse incisors. Anti-mouse CD90.2-FITC (eBioscience 11-0903, 1:50 for staining on sections and 1:400 for cells), Anti-rabbit Celsr1 antibody (Millipore ABT119, 1:200), Anti-rabbit Ki67 antibody (Abcam ab15580, 1:100), Anti-rabbit Phospho Histone 3 (EMD Millipore, 06-570, 1:100), Anti-mouse Phospho Histone 3 (Abcam, ab14955, 1:300), Anti-chicken GFP (Abcam, ab13970, 1:1000). Secondary antibodies used 1:250 for immuostaining on sections and 1:400 for FACS analysis: goat anti-rabbit IgG (H + L) Alexa Fluor 635 (Life technologies, A31576), goat anti-chicken IgG (H + L) Alexa Fluor 488 (Life technologies, A11039), goat anti-mouse IgG (H + L) Alexa Fluor 647 (Life technologies, A21235), rabbit anti-mouse IgG (H + L) Alexa Fluor 546 (Life technologies, A11060), goat anti-mouse IgG (H + L) Alexa Fluor 633 (Life technologies, A21052), donkey anti-rabbit IgG (H + L) Alexa Fluor 594 (Life technologies, A21207), Goat anti-rabbit IgG (H + L) Alexa Fluor 488 (Life technologies, A11008). Hoechst 33342 (Invitrogen 62249, 1:500) was used for DNA staining. Slides were then mounted using Citifluor™ AF1 (Citifluor Ltd., AF1-100) and cover-slipped for microscopy. CD90/Thy1 cre: Confetti incisors were sectioned to 20–30 µm and nuclei were stained with TO-PRO 3-Iodide (Thermo Fisher Scientific, T3605).

An Z., Sabalic M., Bloomquist R.F., Fowler T.E., Streelman T, & Sharpe P.T. (2018). A quiescent cell population replenishes mesenchymal stem cells to drive accelerated growth in mouse incisors. Nature Communications, 9, 378.

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Antibody Reagents for Protein Detection

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The Rabbit polyclonal antibody (pAb) against RAB40C was developed as previously described (Tan et al). The monoclonal antibody (mAb) against AIP-1 (F-3, ^#sc-3565921) (also, DAB2IP) and against c-Myc (9E10, ^#sc-40) were from SANTA CRUZ (Delaware Avenue, CA). mAb against HA-tag (C5^# AG-HT301) were obtained from Trans-Gene® (Beijing, China). Secondary antibodies conjugated with AlexaFluor488, AlexaFluor568 and AlexaFluor635 were purchased from Life Technologies^™ (Thermo Fisher Scientific, Waltham, USA). HRP-conjugated secondary antibodies were purchased from Rockland (Philadelphia, USA).

Luo X., Li C., Tan R., Xu X., Wu W.K., Satoh A., Wang T, & Yu S. (2017). A RasGAP, DAB2IP, regulates lipid droplet homeostasis by serving as GAP toward RAB40C. Oncotarget, 8(49), 85415-85427.

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Immunofluorescence Labeling of Synaptic and Cytoskeletal Proteins

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Anti-synaptotagmin2 (znp1) (Developmental Studies Hybridoma Bank), monoclonal mouse IgG2a, used at 1:300.

Conjugated α-bungarotoxin-AlexaFluor 594 (ThermoFischer Scientific), α-subunit of the nicotinic acetylcholine receptor (AChR) extracted from Bungarus multicinctus venom and conjugated with Alexa Fluor 594 used at 10μg/ml.

Anti-acetylated tubulin clone 6–11-B-1 (Sigma) purified mouse monoclonal IgG antibody 1,5 mg/ml used at 1:200.

Anti-GFP (GeneTex, Euromedex) purified chicken polyclonal IgG antibody, 10mg1ml, used at 1:300.

Goat anti-chicken Alexa Fluor 488 (Life Technologies) purified goat antibody, used at 1:1000

Goat anti-mouse Alexa Fluor 488 (Life Technologies) purified goat antibody, used at 1:1000

Goat anti-mouse Alexa Fluor 635 (Life Technologies) purified goat antibody, used at 1:1000

Bercier V., Hubbard J.M., Fidelin K., Duroure K., Auer T.O., Revenu C., Wyart C, & Del Bene F. (2019). Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities. Molecular Neurodegeneration, 14, 27.

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