Donkey anti rabbit cy3

Manufactured by Jackson ImmunoResearch

Sourced in United States, Panama, United Kingdom, Cameroon

Donkey anti-rabbit Cy3 is a secondary antibody labeled with the fluorescent dye Cy3. It is designed to detect and visualize primary antibodies raised in rabbit in various immunoassays.

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Anti-rabbit Cy3 (101 citations)

136 protocols using donkey anti rabbit cy3

Immunostaining and Imaging Yolk Sacs and BMDMs

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Yolk sacs were stained as described in Newton et al. [33 (link)]. Fixed tissues were stained with rat anti-PECAM-1 (BD, 550274) and rabbit anti-cleaved caspase-3 (CST, 9661). The following secondary antibodies were used: donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 711-165-152) and donkey anti-rat Cy5 (Jackson ImmunoResearch, 712-175-153). Processed yolk sacs were mounted using ProLong Gold Antifade Mountant with DAPI (Invitrogen, P36392) and images were acquired using a LEICA SPE upright confocal microscope with 20x objective. On average about 50 optical sections were collected, representing about 50 μm deep volume, with 1.19 μm step size. The images shown are maximum intensity projections.
BMDMs were seeded at d5 on 4 chamber tissue culture treated glass slides (Falcon, 354104) and treated the following day. The cells were fixed in 4% PFA for 30 min at room temperature. Permeabilized in 0.25% Triton X-100 (w/v) and blocked in 5% BSA (w/v) and 0.05% Triton X-100 (w/v). Staining was performed with p65 (CST, 8242) for 2 h at room temperature followed by labeling with donkey anti-rabbit Cy3 (Jackson ImmunoResearch, 711-165-152) and 1:2000 Hoechst (H3569). The cells were mounted with ProLong Glass Antifade Mountant (P36980). Images were acquired using a LEICA SP8 inverted microscope with a 40x objective.

Kist M., Kőműves L.G., Goncharov T., Dugger D.L., Yu C., Roose-Girma M., Newton K., Webster J.D, & Vucic D. (2020). Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death. Cell Death and Differentiation, 28(3), 985-1000.

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Immunostaining Procedure for Zebrafish Embryos

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Immunostaining was performed as previously described (Lewellis et al., 2013 (link)). Briefly, embryos were fixed in 4% PFA for two hours at room temperature. Following fixation, they were permeabilized in methanol at −20°C, rehydrated, permeabilized with 10 μg/ml proteinase K in PBST for 8 minutes at room temperature, and post-fixed in 4% PFA for 20 minutes at room temperature. Embryos were blocked in 2% BSA in PBST for 1 hour and incubated in primary antibody at 4 degree C overnight. Following PBST washes, they were incubated in secondary antibody overnight at 4 degree C. Rabbit anti-GFP (1:1000, Invitrogen), affinity-purified goat anti-GFP (1:100, (Venkiteswaran et al., 2013 (link))), affinity-purified sheep anti-mCherry (1:1000, custom-made antibody generated against bacterially-produced, recombinant full-length mCherry protein by Covance), zns-2 (1:50, Developmental Studies Hybridoma Bank) and mouse anti-ZO-1 (1:500, ThermoFisher) were detected with goat anti-rabbit-Alexa488 (1:1000, Invitrogen), donkey anti-rabbit- Cy3 (1:1000, Jackson ImmunoResearch), donkey anti-goat-Alexa488 (1:1000, Invitrogen), donkey anti-goat-Cy3 (1:1000, Jackson ImmunoResearch), donkey anti-sheep-Alexa647 (1:1000, Jackson ImmunoResearch), donkey anti-mouse-Cy3 (1:1000, Jackson ImmunoResearch) and donkey anti-mouse Alexa647 (1:1000, Jackson ImmunoResearch) secondary antibodies.

Wang J., Yin Y., Lau S., Sankaran J., Rothenberg E., Wohland T., Meier-Schellersheim M, & Knaut H. (2018). Anosmin1 shuttles Fgf to facilitate its diffusion, increase its local concentration and induce sensory organs. Developmental cell, 46(6), 751-766.e12.

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Immunofluorescence Analysis of Neuromuscular Junctions

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Gastrocnemius muscle was dissected from perfused mice and prepared as described in the immunofluorescence section. Floating 40 μm thick longitudinal sections of gastrocnemius were incubated in a blocking solution containing PBS1x, 0.5% Tween-20, 1.5% BSA for 4 hours at room temperature and then in PBS1x, 0.3% Triton X-100 overnight at room temperature with the polyclonal rabbit anti-synaptophysin antibody at 1:50 (Invitrogen). The sections were washed with PBS1x and then incubated first with donkey anti-rabbit Cy3 (Jackson ImmunoResearch) and α-bungarotoxin-Alexa488 (Invitrogen) at 1:500 for 1 hour at room temperature and then with Fluoromyelin red (Invitrogen) at 1:300 for 30 min. The sections were further washed with PBS1x and mounted. Analysis was performed on a Nikon Eclipse laser scanning confocal microscope. A total of approximately 1,000 neuromuscular junctions were counted from at least 10 sections of gastrocnemius. Individual NMJs were considered as innervated when synaptophysin staining covered at least 50% of the area of α-bungarotoxin staining.

López-Erauskin J., Tadokoro T., Baughn M.W., Myers B., McAlonis-Downes M., Chillon-Marinas C., Asiaban J.N., Artates J., Bui A.T., Vetto A.P., Lee S.K., Le A.V., Sun Y., Jambeau M., Boubaker J., Swing D., Qiu J., Hicks G.G., Ouyang Z., Fu X.D., Tessarollo L., Ling S.C., Parone P.A., Shaw C.E., Marsala M., Lagier-Tourenne C., Cleveland D.W, & Da Cruz S. (2018). ALS/FTD-Linked Mutation in FUS Suppresses Intra-axonal Protein Synthesis and Drives Disease Without Nuclear Loss-of-Function of FUS. Neuron, 100(4), 816-830.e7.

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Immunocytochemical Staining Protocol

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The cells were washed with DPBS and fixed at room temperature for 15 min with 4% paraformaldehyde (Merck Millipore), and washed with DPBS. The fixed cells were blocked for a minimum of 30 min in a blocking solution consisting of KPBS with 0.25% triton-X100 (Fisher Scientific) and 5% donkey serum. The primary antibody (rabbit anti-FOXG1, 1:50 dilution, Abcam, RRID: AB_732415; mouse anti-OCT3/4, 1:500, Santa Cruz Biotechnology, RRID: AB_628051; mouse anti-MAP2, 1:1000, Abcam, RRID: AB_2138153; rabbit anti-PAX6, 1:1000, Biolegend, RRID: AB_2565003; mouse anti-NEUN, 1:1000 dilution, Millipore, RRID: AB_2298772; rabbit anti-TBR1, 1:500, Abcam, RRID: AB_2200219) was added and incubated at room temperature overnight. On the following day, the cells were washed with KPBS and blocked for at least 10 min in donkey serum blocking solution. The secondary antibody (donkey anti-rabbit Cy3, donkey anti-rabbit Cy2, donkey anti-mouse Cy2; 1:200; Jackson Lab) was added with the nuclear stain DAPI (1:1000; Sigma-Aldrich) and incubated at room temperature for approximately 1 h, followed by 2–3 rinses with KPBS. The immunocytochemically labelled cells were then visualized with a Leica microscope (model DMI6000 B), and images were cropped and adjusted in Adobe Photoshop CC 2015.

Grassi D.A., Brattås P.L., Jönsson M.E., Atacho D., Karlsson O., Nolbrant S., Parmar M, & Jakobsson J. (2019). Profiling of lincRNAs in human pluripotent stem cell derived forebrain neural progenitor cells. Heliyon, 6(1), e03067.

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Immunohistochemical Analysis of Retinal Cell Types

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Eyes were enucleated, and retinae were dissected and fixed in 4% formaldehyde for 30 min at room temperature. Fixed retinae were cryoprotected in sucrose in PBS for a few hours and embedded in optical cutting temperature compound on dry ice. Sections (20 μm thick) were cut on a cryostat (Leica). Retinal sections or whole retinal cups were blocked in 5% BSA in PBST (PBS with 0.1% Triton X-100), stained with primary antibodies at 4 °C overnight, and washed three times with PBST. Primary antibodies used in this study included rabbit anti-red/green opsin (1:300, AB5405; EMD Millipore); goat anti-blue opsin (1:100, sc-14365; Santa Cruz Biotechnology Inc.); rabbit anti-GFAP (1:500, Z0344; DAKO); rabbit anti-Iba1 (1:1,000, PA5-21274; ThermoFisher), and rhodamine-conjugated and FITC-conjugated PNA (1:1,000; Vector Laboratories). Sections were stained using secondary antibodies, including donkey anti- rabbit CY3, donkey anti-rabbit Alexa Fluor 647, and donkey anti-goat Alexa Fluor 647 (all used at 1:1,000; Jackson ImmunoResearch), and were costained with DAPI in the dark for 2 h at room temperature and mounted in Fluoromount-G (SouthernBiotech). Images were taken using a 40× objective with Z-stacks on a Zeiss LSM780 confocal microscope. Images used for comparison between groups were taken side by side at the same confocal settings.

Xiong W., Wu D.M., Xue Y., Wang S.K., Chung M.J., Ji X., Rana P., Zhao S.R., Mai S, & Cepko C.L. (2019). AAV cis-regulatory sequences are correlated with ocular toxicity. Proceedings of the National Academy of Sciences of the United States of America, 116(12), 5785-5794.

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Spinal Cord Immunofluorescence of SMA

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Fresh frozen SMA type I (7 months old male) and control patient (3 years old male) spinal cord tissue was a kind gift from Dr. Wandy Chang and the Brain Center at Columbia University respectively. Spinal cords were cut on a cryostat (20 μm thickness). Sections were fixed for 2 minutes in 4% PFA and washed with PBS. Sections were then treated as described above and incubated overnight with antibodies against ChAT - goat polyclonal (1:100; Millipore, AB144P) and rabbit polyclonal anti-human C1q (1:500; Dako, A013602–1). Sections were washed and further incubated with the secondary antibodies: Alexa Fluor 488 donkey anti-goat (Jackson ImmunoResearch, 705–545-003) and Donkey anti-rabbit-Cy3 (Jackson ImmunoResearch, 711–165-152). Sections were washed and mounted in 30% glycerol in PBS.

Vukojicic A., Delestrée N., Fletcher E.V., Pagiazitis J.G., Sankaranarayanan S., Yednock T.A., Barres B.A, & Mentis G.Z. (2019). The Classical Complement Pathway Mediates Microglia-Dependent Remodeling of Spinal Motor Circuits during Development and in SMA. Cell reports, 29(10), 3087-3100.e7.

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Immunostaining of Brain Sections

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Immunostaining was done on 60-μm free-floating coronal sections. Antibodies were applied in tris-buffered saline (TBS) with 3% donkey serum and 0.25% Triton X-100. Immunofluorescence was performed using anti GFP (rabbit polyclonal; 1:500; Invitrogen), anti NeuN (mouse monoclonal; 1:50; a gift from F.H. Gage, Salk Institute for Biological Studies, La Jolla, CA, United States), donkey anti-rabbit Cy3 and donkey anti-mouse Cy5 antibodies (1:250; Jackson Immuno Research Laboratories).

Trinchero M.F., Herrero M, & Schinder A.F. (2019). Rejuvenating the Brain With Chronic Exercise Through Adult Neurogenesis. Frontiers in Neuroscience, 13, 1000.

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Immunohistochemical Analysis of Pig Brain Sections

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Pig brains were aligned on AC-PC prior to sectioning to facilitate comparison with MR images, and cut into 100 μm coronal sections using a Leica SM2500 microtome (Leica microsystems, Milton Keynes, UK). In order to assess the toxicity, 6 sections anterior and posterior to the catheter track were assessed against the control infusions of aCSF. Sections were mounted on gelatine-subbed slides and subsequently subjected to standard immunofluorescent protocols in order to in order to identify neuronal disruption, gliosis and increased macrophage activation.
The sections were incubated overnight at 4°C with either mouse anti-NeuN (1:100; Millipore, CA, USA), rabbit anti-GFAP (1:300; Millipore, CA, USA) or CD63 (1:100; Serotec, Oxfrod UK). Detection was determined using Donkey Anti-Mouse (1:150; streptavidin Alexa Fluor 488) or donkey anti-rabbit Cy3 (1:300; Jackson Laboratories, Sacramento, CA, USA) or Donkey Anti-Mouset Alexa Fluor 488 (1:300, Jackson Laboratories, Sacramento, CA, USA). Sections were also treated with DAPI (1:200 of 1mg/mL; Sigma Aldrich, Dorset, UK) prior to mounting in Fluorsave Reagent before visualisation. Images were captured as described earlier.

Arshad A., Yang B., Bienemann A.S., Barua N.U., Wyatt M.J., Woolley M., Johnson D.E., Edler K.J, & Gill S.S. (2015). Convection-Enhanced Delivery of Carboplatin PLGA Nanoparticles for the Treatment of Glioblastoma. PLoS ONE, 10(7), e0132266.

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Immunohistochemical Analysis of Parvalbumin and CRF

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Mice were anesthetized with choral hydrate and perfused with saline followed by 4% paraformaldehyde in 0.1M phosphate-buffered saline (PBS). The brains were removed, fixed in 4% paraformaldehyde overnight, and subjected to dehydration in increasing saccharin solutions (20–30%) at 4^ºC. The frozen coronal slices of 20 μm thicknesses were prepared and stored at -20^ºC in 20% ethanediol PBS solution containing 20% saccharin. Brain sections were incubated in 3% normal goat serum and 0.2% Triton-X for 1h. Then they were incubated in rabbit anti-Parvalbumin (Swant, Bellinzona) or mouse anti–corticotropin-releasing factor (CRF; Abcam) antibody overnight at 4ºC. Slices were rinsed in PBS then incubated in donkey anti-rabbit Cy3, Alex488, DyLight 647, or donkey anti-mouse Cy3 (Jackson Immunoresearch) for 1h and 4',6-diamidino-2-phenylindole (DAPI) for 10min, then mounted after rinsing. Images were acquired on a microscope using a 20× air objective, a 40× objective (IX-83; Olympus) or a 63× oil immersion objective (Zeiss 510; Carl Zeiss Jena).

Wang L., Shen M., Jiang C., Ma L, & Wang F. (2016). Parvalbumin Interneurons of Central Amygdala Regulate the Negative Affective States and the Expression of Corticotrophin-Releasing Hormone During Morphine Withdrawal. International Journal of Neuropsychopharmacology, 19(11), pyw060.

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Dual Fluorescent Staining of Fish Fins

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Fish fins were frozen in Tissue-Tek O.C.T compound (Sakura Finetek USA, Torrance, CA) and sectioned at 6 microns on a cryostat. Sections were fixed with a mixture of 3.7% paraformaldehyde, 10% Sucrose for 5 minutes at room temperature. Fins were rehydrated with PBS and blocked with 10% normal donkey serum for 1 hour (Jackson Immunoresearch, West Grove, PA). Primary antibody to DsRed (1:300) (Clontech, Mountain View, CA) was added for 1 hour, washed off with PBS, then mixed with primary antibody to GFP conjugated to FITC (1:200) (Rockland, Gilbertsville, PA) followed by a second secondary antibody which was donkey anti-rabbit-Cy3 (1:500), (Jackson Immunoresearch, West Grove, PA) was applied for 1 hour. Fins were washed with PBS and cover-slipped with hard set DAPI, 4,6-diamidino-2-phenylindole (Vector Labs, Burlingame, CA). Slides were imaged by confocal fluorescence microscopy (Olympus BX61, FV500, Olympus Optical, Tokyo, Japan).

Lund T.C., Patrinostro X., Kramer A.C., Stadem P., Higgins L., Markowski T.W., Wroblewski M.S., Lidke D.S., Tolar J, & Blazar B.R. (2014). sdf1 Expression Reveals a Source of Perivascular-Derived Mesenchymal Stem Cells in Zebrafish. Stem cells (Dayton, Ohio), 32(10), 2767-2779.

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