Anti rabbit alexa 568

Manufactured by Thermo Fisher Scientific

Sourced in India, Spain, United States

Anti-rabbit ALEXA 568 is a fluorescent dye conjugated to an antibody that specifically binds to rabbit-derived proteins. It is designed for use in immunofluorescence, flow cytometry, and other applications that require the detection of rabbit-derived targets.

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Lab products found in correlation

Anti mouse alexa 488, by Thermo Fisher Scientific (7 mentions) Cryostat, by Leica (3 mentions) Lsm 710 confocal microscope, by Zeiss (3 mentions) Oct medium, by Electron Microscopy Sciences (2 mentions) Cd11b alexa 647 clone m1 70, by Thermo Fisher Scientific (2 mentions) Vgf peptide antibody, by Thermo Fisher Scientific (2 mentions) Sp5 inverted confocal microscope, by Leica (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by PerkinElmer (2 mentions) Fluoromount g, by Southern Biotech (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) Hlmvec, by Lonza (1 mentions) Scrambled sirna, by Merck Group (1 mentions) Ecl kit, by Bio-Rad (1 mentions) Am966, by Apexbio (1 mentions) Lpa1 sirna, by Merck Group (1 mentions) β actin antibody, by Merck Group (1 mentions) Fugene hd, by Promega (1 mentions) Egm 2 medium, by Lonza (1 mentions) Ab2910, by Santa Cruz Biotechnology (1 mentions) Ab76867, by Abcam (1 mentions)

23 protocols using anti rabbit alexa 568

Endothelial Cell Culture and Analysis

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Human lung microvascular endothelial cells (HLMVECs, Lonza) were cultured at 37°C in an atmosphere of 5% CO₂ with EGM-2 medium (Lonza) containing 25 mL FBS (5%), 0.5 mL hEGF, 2.0 mL hFGF-β, 0.5 mL VEGF, 0.5 mL ascorbic acid, 0.2 mL hydrocortisone, 0.5 mL R3-IGF-1, and 0.5 mL gentamycin. Phospho (T18/S19)-MLC, MLC, antibodies, and cell lysis buffer were obtained from Cell Signaling. Phospho (Y658)-VE-cadherin antibody was purchased from Invitrogen. VE-cadherin antibody was from Santa Crus Biotechnology. β-Actin antibody, scrambled siRNA, LPA1 siRNA, and LPA were from Sigma Aldrich. LPA1 antibody was obtained from Proteintech. AM966 was from Apex Bio. Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies, ECL kit, and SDS-PAGE for western blotting were purchased from Bio-Rad Laboratories, Inc. For immunostaining, anti-mouse Alexa-488, anti-rabbit Alexa-568, and DAPI were from Invitrogen. Transfection reagent FuGENE HD was from Promega. All other reagents were of analytical grade.

Cai J., Wei J., Li S., Suber T, & Zhao J. (2017). AM966, an Antagonist of Lysophosphatidic Acid Receptor 1, Increases Lung Microvascular Endothelial Permeability through Activation of Rho Signaling Pathway and Phosphorylation of VE-Cadherin. Mediators of Inflammation, 2017, 6893560.

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Immunofluorescence Staining of Caveolins

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The unroofed cells were incubated in 3% bovine serum albumin/PBS (BSA, m/v, fresh, Fisher Bioreagents #BP9703) for 1.5 h, followed by primary antibody (1:100 in 3%BSA/PBS) incubation for 1 h. Next, cells were washed thoroughly with PBS and the secondary antibody tagged with fluorescence dye (1:500) or GFP-nanobody (1:500) was applied for 1 h. Afterwards, cells were washed 4 times in PBS and stored in fresh PBS at 4 C until the samples were imaged. The following antibodies were used: anti-Caveolin1-Rabbit (abcam #ab2910), anti-Caveolin1-mouse (Santa Cruz #sc-53564), anti-Cavin1-Rabbit (abcam #76919), anti-Cavin2-Rabbit (abcam #ab76867), anti-Cavin3-Rabbit (abcam #abcam2912), anti-EHD2-goat (abcam #ab23935), anti-Pacsin2-Rabbit (Proteintech #10518-2-AP), anti-EHBP1-Rabbit (Proteintech #17637-1-AP), anti-mouse-Clathrin heavy chain (Thermo-Fisher #MA1-065, 1:2000), anti-mouse-Dynamin2 (Santa Cruz, C-18; #sc-6400), anti-rabbit-Atto647N (Rockland #611-156-122), anti-goat-Atto647N (Rockland, #610-156-121), anti-goat-Atto647N (Rockland #605-456-013 S), anti-rabbit-Alexa568 (Invitrogen #A11036), Fab2-anti-rabbit-Alexa594 (ThermoFisher #A-11072), Fab2-anti-mouse-Alexa488 (ThermoFisher #A-11017), Fab2-anti-mouse-Alexa568 (ThermoFisher #A-11019), GFP-nanobody-Atto647N (Chromotek #gba647n-100), Phalloidin-Alexa488 (ThermoFisher #A12379).

Matthaeus C., Sochacki K.A., Dickey A.M., Puchkov D., Haucke V., Lehmann M, & Taraska J.W. (2022). The molecular organization of differentially curved caveolae indicates bendable structural units at the plasma membrane. Nature Communications, 13, 7234.

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Immunohistochemistry for Microglia Assessment

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After BI, GTS-21 or saline-treatment (n = 3), IHC to detect microglia changes was performed using anti-Iba1. ^{20 (link),30 (link)} Rats were anesthetized as described, chest opened, a needle was inserted through the heart and ascending aorta perfused via infusion pump with 200ml of 0.01m phosphate buffered saline (PBS, pH 7.35) followed by 300ml paraformaldehyde in 0.1 M in PBS. Then, spinal segments were excised, post fixed (4% formaldehyde) and kept in sucrose (10%) for 4h, sucrose(20%) for 8h and then sucrose(30%) overnight. After tissue dehydration, spinal segments were paraffin embedded, sectioned with a cryostat (8μm thick), mounted on Shandon™ Polysine slides (Fisher-Scientific, Waltham, MA) and stored at −20^oC. All IHC sections were treated under the same conditions on the same day to minimize the between-group variability. Sections were blocked (1.5% goat serum and 0.04% Saponin in 1% bovine serum albumin) for 1h, incubated overnight at 4^oC with primary antibody, rabbit anti Iba1(1:150, Wako-Pure Industries, Richmond, VA) and incubated for 90min with anti-rabbit Alexa 568 (1:300; Invitrogen, Grand Island). Randomly-selected IHC images were scanned using a confocal microscope (Zeiss LSM 800; Carl Zeiss Microscopy, Thornwood, NY). Photoshop program (Volocity 6.3, Perkin Elmer, Inc) was used for quantitation of expression of Iba1.

Zhou Y., Leung-Pitt Y., Deng H., Ren Y., You Z., Kem W.R., Shen S., Zhang W., Mao J, & Martyn J.A. (2021). Non-opioid, GTS-21 Mitigates Burn Injury Pain in Rats by Decreasing Spinal Cord Inflammatory Responses. Anesthesia and analgesia, 132(1), 240-252.

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Immunostaining of Golgi Apparatus

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For immunofluorescence with anti-giantin antibody, cells were fixed in 4% paraformaldehyde for 20 min at r.t., washed twice in PBS and permeabilized with 0.05% saponin in PEM buffer (80 mM PIPES, 5 mM EGTA, 1 mM MgCl₂pH 6.8) for 5 min. Incubation with anti-giantin at 1∶500 dilution was carried out in PBS containing 0.05% saponin for 1 h. After PBS washes, cells were incubated with anti-rabbit-Alexa 568 (Invitrogen) at 1∶200 in 1% BSA in PBS for a further hour.

Oeste C.L., Pinar M., Schink K.O., Martínez-Turrión J., Stenmark H., Peñalva M.A, & Pérez-Sala D. (2014). An Isoprenylation and Palmitoylation Motif Promotes Intraluminal Vesicle Delivery of Proteins in Cells from Distant Species. PLoS ONE, 9(9), e107190.

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Immunohistochemical Analysis of Ear Tissue

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Ears were fixed in periodate-lysine-paraformaldehyde overnight as reported 49 (link), cryoprotected in 15% sucrose, embedded in OCT medium (Electron Microscopy Sciences) and frozen in dry-ice cooled isopentane. Eighteen-micron cross-sections were cut on a Leica cryostat (Leica Microsystems), blocked with 5% goat or donkey serum, then stained with a combination of DAPI (Perkin Elmer), CD11b-Alexa 647 (clone M1/70, eBioscience), and VGF peptide antibody followed by anti-rabbit Alexa 568 (Invitrogen). Images were acquired on an inverted SP5 confocal microscope (Leica Microsystems) using identical PMT (photomultiplier tube) and laser settings. Lesion depth was scored manually using Imaris software.

Beerli C., Yakimovich A., Kilcher S., Reynoso G.V., Fläschner G., Müller D.J., Hickman H.D, & Mercer J. (2018). Vaccinia virus hijacks EGFR signalling to enhance virus spread through rapid and directed infected cell motility. Nature microbiology, 4(2), 216-225.

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Immunohistochemical Analysis of Ear Tissue

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Immunofluorescence Staining of Golgi and Membrane Proteins

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Cells were grown on glass coverslips for one day, then fixed with 3% PFA for 10 min, quenched with 50 mM NH4Cl in PBS for 5 min, permeabilized with 0.2% TritonX-100 in PBS for 10 min, blocked with 1% BSA in PBS for 1 h, and incubated with primary antibodies diluted in 1% BSA in PBS for 1 h: Anti-AP1g1 (1:1000, self-made from hybridoma cells), anti-bCOP (1:500, CM1, hybridoma supernatant, gift from Dr. Felix Wieland, Heidelberg University), anti-GGA2 (1:500, BD Bioscience 612613), anti-GM130 (1:1000, Cell Signaling 12480S), and anti-TGN46 (1:1000, BioRad AHP500G). Samples were washed and incubated with fluorescent secondary antibodies diluted in 1% BSA in PBS for 1 h (1:400; anti-mouse-Alexa488, Invitrogen A21202; anti-rabbit-Alexa568, Invitrogen A10042; anti-sheep-Cy3, Jackson ImmunoResearch 713-165-147). Coverslips were mounted in FluoromountG (SouthernBiotech) supplemented with 0.5 ng/ml DAPI (Sigma-Aldrich) and stored in the dark at 4°C. For localization of GFP-KDEL, cells were grown on coverslips for one day, transfected with the pcDNA3-ss-GFP-KDEL, and fixed and stained a day later.

Pennauer M., Buczak K., Prescianotto‐Baschong C., & Spiess M. (2021). Shared and specific functions of Arfs 1–5 at the Golgi revealed by systematic knockouts.

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Regulatory T Cell Phenotyping

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Colon sections were cut 5 mm, and after heat-induced epitope retravel, cells were incubated overnight at 4°C with rabbit anti-human FoxP3 polyclonal antibody (Abcam). After washing, sections were stained with a secondary antibody anti-rabbit Alexa 568 (ThermoFisher) for two hours at room temperature. The sections were washed and incubated for two hours with the following antibodies: anti-human mouse CD4 (clone: RPA-T4), CD39 (clone: TU66), CD25 (clone: M-A251), and CD73 (clone: AD2) (BD Bioscience). Afterward, the sections were incubated with DAPI and mounted on slides using ProLong Gold antifade reagent (ThermoFisher). Images were acquired on the LSM 710 confocal microscope (Carl Zeiss) with a 20X objective. The colocalization was assessed using Zen 2012 SP2. The expression of CD4⁺CD25⁺FoxP3⁺CD39⁺CD73⁺ defined the Treg suppressor; for the Treg cells, the expression was used CD4⁺CD25⁺FoxP3⁺. At least ten different fields were counted for each sample.

Rosseto-Welter E.A., D'argenio-Garcia L., Blasco Tavares Pereira Lopes F., Zulim Carvalho A.E., Flaquer F., Severo-Lemos V., Viero Nora C.C., Steinwurz F., Pires Garcia Oliveria L., Aloia T., Rizzo L.V., Pitangueira Mangueira C.L, & Carvalho K.I. (2022). Biologic Agents in Crohn's Patients Reduce CD4+ T Cells Activation and Are Inversely Related to Treg Cells. Canadian Journal of Gastroenterology & Hepatology, 2022, 1307159.

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Immunofluorescence Staining of Cadherin-11 in Fibroblast-like Synoviocytes

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Fibroblast-like synoviocytes were plated overnight in wells of a 24-well plate, fixed with Zamboni's fixative^{57 (link)} for 10 minutes, permeabilized with 0.05% Triton X-100, and blocked with antibody diluent (0.2% [vol/vol] Triton X-100, 5% [vol/vol] donkey serum, and 1% [vol/vol] bovine serum albumin in PBS) for 30 minutes. The cells were then incubated overnight at 4°C in 1:100 (in antibody diluent) anti-cadherin-11 antibody (CDH-11, rabbit polyclonal, Thermo Fisher, 71-7600). Cells were washed 3 times with PBS-Tween and incubated in the conjugated secondary antibody, anti-rabbit Alexa-568 (1:1000 in PBS, Thermo Fisher, A10042), for 1 hour at room temperature (21°C). The secondary antibody was washed off 3 times with PBS-Tween and the cells were incubated in the nuclear dye DAPI (1:1000 in PBS, Sigma, D9452) for 10 minutes. Cells were further washed with PBS-Tween once and imaged in PBS using an EVOS FLoid Cell Imaging Station (Thermo Fisher) at 598 nm (for CDH-11) and 350 nm (for DAPI) wavelengths of light. Cells without primary antibody did not show fluorescence.

Chakrabarti S., Hore Z., Pattison L.A., Lalnunhlimi S., Bhebhe C.N., Callejo G., Bulmer D.C., Taams L.S., Denk F, & Smith E.S. (2020). Sensitization of knee-innervating sensory neurons by tumor necrosis factor-α-activated fibroblast-like synoviocytes: an in vitro, coculture model of inflammatory pain. Pain, 161(9), 2129-2141.

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Modulation of FGFR3 Phosphorylation

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MH7A cells (1 × 10⁴ cells/cm²) were seeded into four-chamber culture slides and cultured for 12 h. The cells were then starved with FBS-free MEM for 16 h, pretreated with the indicated doses of kaempferol, U0126, and PKC412 for 30 min, and then co-treated with bFGF (10 ng/ml). The cells were fixed with 4% formalin, permeabilized with 0.5% Triton X-100/1 × PBS, blocked with 1% of BSA, and hybridized with anti-rabbit phospho-FGFR3 Tyr724-specific antibodies overnight at 4 °C in a humidified chamber. The FGFR3 proteins were visualized by hybridization with secondary antibodies conjugated with anti-rabbit-Alexa-568 (Cat#: A11036, Thermo Fisher Scientific) under an ECLIPSE Ti inverted fluorescence microscope (NIKON Instruments Korea).

Lee C.J., Moon S.J., Jeong J.H., Lee S., Lee M.H., Yoo S.M., Lee H.S., Kang H.C., Lee J.Y., Lee W.S., Lee H.J., Kim E.K., Jhun J.Y., Cho M.L., Min J.K, & Cho Y.Y. (2018). Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis. Cell Death & Disease, 9(3), 401.

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