Goat anti rabbit alexafluor546

Manufactured by Thermo Fisher Scientific

Sourced in United States

Goat anti-rabbit AlexaFluor546 is a secondary antibody conjugated with the AlexaFluor546 fluorescent dye. It is designed to bind and detect rabbit primary antibodies in various immunoassay and imaging applications.

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8 protocols using goat anti rabbit alexafluor546

Immunofluorescent Localization of Slack Channel

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CHO cells were plated onto glass coverslips and transiently transfected, as described above. Approximately 36 hours after transfection cells were fixed for 10 minutes in 4% paraformaldehyde. Membranes were then permeablized for 5 minutes in 1X PBS containing 0.2% Triton X-100. Following permeablization and a wash in 1XPBS, cells were incubated for 1 hour in 3% BSA and Rabbit Anti-Slack (1:500). Cells were again washed in 1XPBS before a 20 minute incubation with secondary antibody Goat Anti-Rabbit AlexaFluor 546 (1:1000, Molecular Probes). After a last washing step in 1X PBS, coverslips were mounted onto glass microscope slides with dapi containing VectaShield mounting medium (Vector Laboratories).

Evely K.M., Pryce K.D, & Bhattacharjee A. (2017). The Phe932Ile mutation in KCNT1 channels associated with severe epilepsy, delayed myelination and leukoencephalopathy produces a loss-of-function channel phenotype. Neuroscience, 351, 65-70.

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Quantifying DNA Double-Strand Breaks via γH2AX Foci

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The number of γH2AX foci was used as a biomarker for DSBs at two different time points after irradiation with 2 and 4 Gy, respectively. Following fixation with 4% paraformaldehyde for 15 min at room temperature, cells were permeabilized with ice cold methanol for 30 min. Cells were then blocked with 20% fetal calf serum in phosphate-buffered saline (PBS). Immunostaining was performed for 1 h at room temperature using the primary rabbit polyclonal anti-γH2AX antibody (pS139, ab11174, Abcam, Cambridge, UK), diluted 1:5000 with PBS, followed by three washing steps in PBS. Secondary staining was performed for 30 min in the dark with goat anti-rabbit Alexa-Fluor 546 (Molecular Probes, Eugene, OR, USA), diluted 1:1000. After washing three times with PBS, preparations were mounted with Vectashield Mounting Medium containing DAPI (Vector Laboratories, Burlingame, USA). Fifty cells per dose and time point were evaluated using a Zeiss Axioskop fluorescence microscope (Zeiss, Oberkochen, Germany).

Maierhofer A., Flunkert J., Dittrich M., Müller T., Schindler D., Nanda I, & Haaf T. (2017). Analysis of global DNA methylation changes in primary human fibroblasts in the early phase following X-ray irradiation. PLoS ONE, 12(5), e0177442.

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Antibody Validation for Western Blot and IF

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Antibodies for Western blot and immunofluorescence staining were purchased as follows: PAK1, PAK2, PAK3, PAK4, phospho-PAK4/5/6 (S474, S602, S560), PARP1, ATM, DNA-PK, PAK5, phospho-DNA PKs (S2056), and PAK6 (all Cell Signaling Technology, Frankfurt am Main, Germany); β-actin (Sigma-Aldrich); phospho-ATM (S1981) (Rockland Immunochemicals, Gilbertsville, PA, USA); Rad51 and γH2AX (both Merck Millipore, Burlington, MA, USA), 53BP1 (Novus Biologicals, Wiesbaden Nordenstadt, Germany); goat anti-mouse AlexaFluor488 and goat anti-rabbit AlexaFluor546 (both Life Technologies, Carlsbad, CA, USA); donkey anti-rabbit HRP and sheep anti-mouse HRP (both GE Healthcare, Chicago, IL, USA).

Blankenstein L.J., Cordes N., Kunz-Schughart L.A, & Vehlow A. (2022). Targeting of p21-Activated Kinase 4 Radiosensitizes Glioblastoma Cells via Impaired DNA Repair. Cells, 11(14), 2133.

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Neuromuscular Junction Histology Protocol

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The left gastrocnemius muscles were frozen in OCT floated on liquid nitrogen and stored at −80°C until sectioning. Samples were acclimated 30min in the cryostat and sectioned at −20°C in 50μm mid-belly longitudinal sections onto Superfrost Plus glass slides (Fisher Scientific). Slides were dried 1hr at room temperature (RT) and stored at −20°C until histological processing. At RT, slides were thawed 30min, fixed in 4% PFA 30min, and washed (3×10min) in 1X PBS. Slides were incubated in blocking solution (10% goat serum, 4% BSA, 3% Triton-X-100, in 1X PBS) 2hr at RT, followed by overnight incubation at 4°C in primary antibody solution containing chicken α-NF-200 (Abcam, Ab72996, [1:5000]) and rabbit α-Synapsin-1 (Cell Signaling, 5297S, [1:200]). After washes (3×10min) in 1X PBS, slides were incubated 2hr at RT in secondary antibody solution containing goat anti-Chicken Alexa Fluor 594 (Life Technologies, A11042, [1:1000]), goat anti-Rabbit Alexa Fluor 546 (Life Technologies, A11010, [1:1000]), and α-bungarotoxin (-BTX) Alexa Fluor 488 (Life Technologies, B13422, [1:1000]). Slides were then washed (3×10min) in 1X PBS and mounted with Fluoromount-G (Invitrogen).

Balch M.H., Harris H., Chugh D., Gnyawali S., Rink C., Nimjee S.M, & Arnold W.D. (2021). Ischemic Stroke-Induced Polyaxonal Innervation at the Neuromuscular Junction is Attenuated By Robot-Assisted Mechanical Therapy. Experimental neurology, 343, 113767.

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Immunohistochemical Staining of Brain Sections

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The experimental procedure was based on previously published protocols (Cisbani et al., 2013 (link); Cicchetti et al., 2014 (link)). In brief, prior to immunostaining, sections were deparaffinized in Citrisolv™ (Fisherbrand), rehydrated and incubated overnight at room temperature in blocking solutions containing the primary antibodies listed in Supplementary Table 1. After KPBS washes, sections were incubated with appropriate secondary antibodies in a blocking solution for 2 h at room temperature (Goat anti-mouse Alexa Fluor® 488, 1:750; Goat anti-rabbit Alexa Fluor® 546, 1:500; Goat anti-chicken Alexa Fluor® 647, 1:500; all Life Technologies).
Following DAPI incubation (2 mg/ml, Molecular Probes), sections were washed and coverslipped with Fluoromount-G™ (SouthernBiotech). Confocal laser scanning microscopy was performed using an Olympus FV500 confocal laser-scanning microscope. Images were acquired by sequential scanning and optimized by a two-frame Kalman filter and analysed using acquisition software from Olympus (Fluoview SV500 imaging software 4.3, Olympus) and ImageJ (NIH).

Vuono R., Winder-Rhodes S., de Silva R., Cisbani G., Drouin-Ouellet J., Spillantini M.G., Cicchetti F, & Barker R.A. (2015). The role of tau in the pathological process and clinical expression of Huntington’s disease. Brain, 138(7), 1907-1918.

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Epidermal Sheet Immunostaining

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Staining of acetone-fixed epidermal sheets was performed with the following primary antibodies: CD1a (mIgG2b; BioLegend, USA), CD83 (BD Biosciences, USA), CD207 (Sigma-Aldrich, USA), and HLA-DR-Alexa88 (mIgG2; BioLegend, USA). Primary antibodies were incubated overnight at 4°C and respective isotype controls were performed. After a washing step, species- and isotype-specific secondary Abs goat anti-mouse Alexa Fluor488 and goat anti-rabbit Alexa Fluor546 (both Life Technologies, USA) were added when needed and incubated for 1 hour at room temperature. Sheets were mounted with 20 μl mounting media containing DAPI (Vector Laboratories Inc., USA). Images were taken using the AX70 microscope with the imaging software MetaMorph version 7.8.6.0 (Olympus, Germany).

Nikolić N., Kienzl P., Tajpara P., Vierhapper M., Matiasek J, & Elbe-Bürger A. (2019). The Antiseptic Octenidine Inhibits Langerhans Cell Activation and Modulates Cytokine Expression upon Superficial Wounding with Tape Stripping. Journal of Immunology Research, 2019, 5143635.

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Immunofluorescence Assay for Organelle Labeling

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IFAs were performed as described previously (van Dooren et al., 2008 (link)). Primary antibodies used were mouse anti-c-myc (1:200 dilution; Santa Cruz clone 9E10), rat anti-HA (1:200 dilution; Sigma clone 3F10), and rabbit anti-Tom40 (1:2000 dilution; van Dooren et al., 2016 (link)). Secondary antibodies used were goat anti-mouse Alexa Fluor 488 (1:500 dilution; Life Technologies), goat anti-rat Alexa Fluor 488 (1:100 to 1:500 dilution; Life Technologies), goat anti-rat CF 488A (1:100 to 1:500 dilution; Sigma), and goat anti-rabbit Alexa Fluor 546 (1:500 dilution; Life Technologies). Biotinylation was performed as outlined for the proteomics, except that mtAPEX samples were incubated in H₂O₂ for 1 min. For visualizing biotinylated proteins, we used Oregon Green-conjugated avidin (1:1000 dilution; Life Technologies). Images were acquired on a DeltaVision Elite deconvolution microscope (GE Healthcare) fitted with a 100X UPlanSApo oil immersion objective lens (NA 1.40). Images were deconvolved and adjusted for contrast and brightness using SoftWoRx Suite 2.0 software, and subsequently processed using Adobe Illustrator.

Seidi A., Muellner-Wong L.S., Rajendran E., Tjhin E.T., Dagley L.F., Aw V.Y., Faou P., Webb A.I., Tonkin C.J, & van Dooren G.G. (2018). Elucidating the mitochondrial proteome of Toxoplasma gondii reveals the presence of a divergent cytochrome c oxidase. eLife, 7, e38131.

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Immunofluorescent Labeling of Cellular Markers

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Cells were fixed for 20 min in methanol at −20°C, washed with PBS and labeled with antibodies as previously described (45 (link)). Briefly, cells were incubated with primary antibodies diluted in PBS for 2 h at room temperature. After a second wash with PBS, cells were incubated with fluorophore-conjugated antibodies for 1 h at room temperature. Antibodies used are mouse anti-dsRNA (J2) (Scicons, Hungary), rabbit polyclonal anti-DOM3Z (Cedarlane), rabbit polyclonal anti-DUSP11 (ProteinTech), goat anti-mouse AlexaFluor 488 (Fisher) and goat anti-rabbit AlexaFluor 546 (LifeTech). Cells were washed in PBS, rinsed in H₂O and dried before being mounted with DAPI (VectaShield, Vector Laboratories Inc.). Cells were visualized with indirect immunofluorescence using a Zeiss AxioObserver inverted microscope and a 63× oil objective. Images were processed using the Zeiss Zen Pro software.

Amador-Cañizares Y., Bernier A., Wilson J.A, & Sagan S.M. (2018). miR-122 does not impact recognition of the HCV genome by innate sensors of RNA but rather protects the 5′ end from the cellular pyrophosphatases, DOM3Z and DUSP11. Nucleic Acids Research, 46(10), 5139-5158.

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