Donkey anti mouse cy3

Manufactured by Jackson ImmunoResearch

Sourced in United States

Donkey anti-mouse Cy3 is a secondary antibody conjugated with the fluorescent dye Cy3. It is designed to detect and visualize mouse primary antibodies in various immunoassays and imaging techniques.

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81 protocols using donkey anti mouse cy3

Visualization of Ovarian Acetylated α-Tubulin

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For the visualization of acetylated α-tubulin, ovaries from 2-day-old well-fed females were dissected into ice-cold methanol and fixed for 20 min at −20 °C. Ovaries were subsequently washed and rehydrated with PBS for 1 h, blocked in 10% goat serum in PBS for 1 h, and stained overnight at 4 °C with mouse anti-acetylated-α-tubulin antibody (1:500). After washing, ovaries were incubated with a secondary antibody donkey anti-mouse-Cy3 (1:500) (Jackson ImmunoResearch 715-165-151) and stained with DAPI. For visualization of Me31B, dGe1, and DCP1, ovaries were dissected into 2% v/v PFA (Electron Microscopy Sciences, #15710), 0.05% v/v Triton X-100 (Sigma, P1379) in PBS (pH 7.4) and fixed for 20 min. After washes in PBST (0.1% Tween20 in PBS), ovaries were permeabilized in 1% Triton X-100 in PBS for 1 h, blocked with 10% goat serum in PBS and stained with primary antibodies (1:500) in blocking buffer. After washing, ovaries were incubated with a secondary antibody, donkey anti-mouse-Cy3 (1:500) (Jackson ImmunoResearch, 715-165-151) for Me31B and dGe1, and anti-GFP antibody-Alexa Fluor 488 conjugate (1:500) (Invitrogen, A-21311) for YFP-DCP1. The samples were then stained with DAPI.
Stacks of images were acquired on a Leica TCS SP8 LIGHTNING confocal microscope using a 63 × 1.4 NA oil immersion objective. Images were processed and analyzed using ImageJ (http://rsb.info.nih.gov/ij/).

Wippich F., V.a.i.s.h.a.l.i., Hennrich M.L, & Ephrussi A. (2023). Nutritional stress-induced regulation of microtubule organization and mRNP transport by HDAC1 controlled α-tubulin acetylation. Communications Biology, 6, 776.

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Immunofluorescence Imaging of Adipose Tissue

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Immunofluorescence was performed on 5–8 μm cryostat sections of tissues freshly embedded in OCT as described^{29 (link)}. The sections were fixed in 4% paraformaldehyde in PBS and then stained with rat anti-PECAM (1:200, BD Biosciences #557355), mouse anti-α smooth muscle actin (1:200, Abcam #ab7817), rabbit anti-phospho-JNK (1:200, Cell Signaling), rabbit anti-UCP1 (1:200, Abcam #ab10983) and rabbit anti-ERα (1:200, Abcam #ab2746) as described^{29 (link)}. Secondary antibodies, used at a 1:500 dilution, included cy3 donkey anti-rabbit, cy3 donkey anti-mouse, cy3 donkey anti-rat, cy5 donkey anti-rat, cy5 donkey anti-rabbit, cy3 donkey anti-mouse were from Jackson ImmunoResearch. Immunostaining images were collected on a Zeiss LSM500 confocal microscope, an Olympus IX70 inverted microscope or an Olympus upright BX40 microscope. Direct GFP and RFP fluorescence for whole-adipose depots were imaged and photographed with a Zeiss Stemi SV11 microscope. Cryostat sectioning was performed with a Microm HM505 E cryostat. For BrdU staining, the cells, sections or tissues were fixed and washed in H₂O, incubated with 1 N HCl at 37 °C for 45 min, washed in H₂O, incubated in 0.1 M NaBO₄ for 10 min and subjected to immunohistochemistry.

Lapid K., Lim A., Clegg D.J., Zeve D, & Graff J.M. (2014). Oestrogen signalling in white adipose progenitor cells inhibits differentiation into brown adipose and smooth muscle cells. Nature communications, 5, 5196.

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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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Phenotyping and ZIKV Infection of pDCs

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The phenotype of pDCs was assessed with the following primary mAbs (BD Horizon): CCR7-FITC (clone 3D12), CD83-PE-Cy7 (clone HB15e), CD86-PE-Cy5 (clone 233), HLA-DR-APC-H7 (clone L243), and CD123-PerCP-Vio 700 (clone AC145). CD303-PE (clone REA693) was purchase from MACS. Cells were fixed with 4% PFA for 20 min, stained for 30 min at 4°C and washed before being subjected to FACS analysis. ZIKV infectivity was assessed with the mouse anti-pan flavivirus envelope protein mAb 4G2 (RD Biotech). A ZIKV-specific polyclonal antibody targeting the envelope protein and the non-structural protein 1 (20 (link)) or the mouse anti-dsRNA IgG2a mAb J2 (Scicons) as indicated. Cells were fixed with 4% PFA for 20 min at room temperature (RT) and permeabilized with 0.1% Triton X-100 in PBS for 4 min at RT. Fixed cells were stained overnight at 4°C using 4G2 or J2 (1:500) in PBS-BSA. Then, cells were stained 20 min at RT with secondary antibody donkey anti-mouse Alexa Fluor 488 IgG (1:1000, Invitrogen) or donkey anti-mouse Cy3 (1:500, Jackson immunoResearch) in PBS-BSA, and washed before being subjected to FACS analysis. At least, 5,000 events were acquired using Cyan cytometer (Beckman Coulter). Stained cells were analyzed using FlowJo software (Tree Star, Inc., Ashaland, OR). pDCs survival was assessed using the 7-AAD assay as previously described (21 (link)).

Bos S., Poirier-Beaudouin B., Seffer V., Manich M., Mardi C., Desprès P., Gadea G, & Gougeon M.L. (2020). Zika Virus Inhibits IFN-α Response by Human Plasmacytoid Dendritic Cells and Induces NS1-Dependent Triggering of CD303 (BDCA-2) Signaling. Frontiers in Immunology, 11, 582061.

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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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Immunofluorescent Labeling of Neuronal Markers

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Sections were incubated in primary antibody in 0.01 M PBS with 5% bovine serum albumin and 0.3% Triton at 4°C overnight. Primary antibody was removed by washing three times for 5 min each in 0.01 M PBS. Sections were then incubated with appropriate secondary antibodies for 2 h at room temperature in 0.01 M PBS with 0.3% triton and 5% normal donkey serum, and then washed in 0.01 M PBS three times for 5 min. Slides were then coverslipped using Mowiol mounting medium. Primary antibodies used were rabbit anti-GFAP (1:2,000, Dako), mouse anti-NeuN (1:500, Millipore), and mouse anti-MAP2 (1:500, Sigma). Secondary antibodies used were donkey anti-rabbit Alexa 488 (Invitrogen), donkey anti-mouse Alexa 546 (Invitrogen), and donkey anti-mouse Cy3 (Jackson ImmunoResearch Inc.). All secondary antibodies were applied at a dilution of 1:1,000.

James N.D., Angéria M., Bradbury E.J., Damberg P., McMahon S.B., Risling M, & Carlstedt T. (2017). Structural and Functional Substitution of Deleted Primary Sensory Neurons by New Growth from Intrinsic Spinal Cord Nerve Cells: An Alternative Concept in Reconstruction of Spinal Cord Circuits. Frontiers in Neurology, 8, 358.

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Immunofluorescent Staining of Mouse Brain Samples

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Mouse brain samples for immunofluorescent staining were prepared as previously reported [20 (link), 21 (link)]. Sections for staining were incubated with the following primary antibodies: rabbit anti Iba1 (1:2000, Wako) and mouse anti-Aβ (6E10, 1:1000, Covance) at 4 °C overnight. After being washing with PBS, the sections were incubated with donkey anti-rabbit Alexa 488 (1:500; Jackson ImmunoResearch) and donkey anti-mouse Cy3 (1:500; Jackson ImmunoResearch) at 4 °C for 2 h. After further washing, the sections were mounted in Vectashield anti-fading medium (Vector Laboratories). The fluorescence images were observed using a confocal laser scanning microscope (CLSM; C2si, Nikon).

Ni J., Wu Z., Meng J., Saito T., Saido T.C., Qing H, & Nakanishi H. (2019). An impaired intrinsic microglial clock system induces neuroinflammatory alterations in the early stage of amyloid precursor protein knock-in mouse brain. Journal of Neuroinflammation, 16, 173.

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Immunohistochemical Localization of Serotonergic Markers

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Immunohistochemistry was performed either on alternate series of sections or in combination with ISH. Sections were washed in PBS, then in PGT (PBS with 0.2% gelatin and 0.25% Triton X-100) four times for 15 min. Sections were incubated overnight at 4°C with the following primary antibodies: anti-Tph2 (mouse monoclonal, 1/1000, Sigma-Aldrich), anti-5-HT (rabbit polyclonal, 1/1000, Sigma-Aldrich), anti–serotonin transporter (anti-SERT; rabbit polyclonal, 1/1000, Calbiochem). For fluorescence microscopy, sections were then incubated for 2 h at room temperature with the following secondary antibodies: donkey anti-rabbit 488 (1/200, The Jackson Laboratory), donkey anti-rabbit Cy3 (1/200, The Jackson Laboratory), donkey anti-mouse 488 (1/200, The Jackson Laboratory), donkey anti-mouse Cy3 (1/200, The Jackson Laboratory), or phalloidin 594 (1/40, Invitrogen). Sections were rinsed in PB, mounted in mowiol-Dabco (25 mg/ml), and stored at 4°C.

Teng T., Gaillard A., Muzerelle A, & Gaspar P. (2017). EphrinA5 Signaling Is Required for the Distinctive Targeting of Raphe Serotonin Neurons in the Forebrain. eNeuro, 4(1), ENEURO.0327-16.2017.

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Quantification of Neutrophil NETs

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Neutrophils seeded on glass coverslips were stained and quantified as previously described (Brinkmann et al., 2012 (link)). Briefly, cells were fixed in 2% paraformaldehyde (PFA) post-NET induction, permeabilized on 0.5% Triton-X100, blocked for 30 min in blocking buffer. Neutrophils were then stained with the following primary antibodies: anti-neutrophil elastase (Calbiochem: 481001, RRID:AB_212213) and antibodies directed against the subnucleosomal complex of Histone 2A, Histone 2B, and chromatin ([Losman et al., 1992 (link)], generated in house). The secondary antibodies donkey anti-mouse Cy3 (Jackson ImmunoResearch Labs: 715-175-150, RRID:AB_2340819) and donkey anti-rabbit Alexa Fluor488 (Life Technologies: A11008, RRID:AB_143165) were used. Finally, the samples were stained with Hoechst 33342 (Immunochemistry: 639, RRID:AB_2651135) and mounted with Mowiol. Image acquisition was c using a Leica DMR upright fluorescence microscope equipped with a Jenoptic B/W digital microscope camera and analysed using ImageJ/FIJI software.

Kenny E.F., Herzig A., Krüger R., Muth A., Mondal S., Thompson P.R., Brinkmann V., von Bernuth H, & Zychlinsky A. (2017). Diverse stimuli engage different neutrophil extracellular trap pathways. eLife, 6, e24437.

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Immunohistochemistry of P-IRE1α in Multiple Sclerosis

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Frozen brain tissue from 4 MS patients and 4 healthy control individuals was cut into 7 μm thick sections, air dried, and fixed in ice-cold acetone for 10 minutes. Sections were delipidised in 70% ethanol for 5 minutes, followed by blocking of non-specific binding with 10% donkey serum (#D9663, Sigma-Aldrich). Rabbit anti-human P-IRE1α (#NB100–2323, Novus Biologicals, 1:50) was incubated with mouse anti-human GFAP-Cy3 (#C9205, Sigma-Aldrich, 1:50), mouse anti-human NeuN (#MAB377, Millipore, 1:50), mouse anti-human CNPase (#MAB326, Millipore, 1:100) or mouse anti-human CD14 (#555397, BD Biosciences, 1:10) in blocking buffer overnight at 4C. The next day slides were washed with 0.05% PBS-Tween and incubated with a mixture of donkey anti-rabbit Alexa Fluor 488 (#R37118, Life Technologies, 1:400) and donkey anti-mouse-Cy3 (#715–165-151, Jackson ImmunoResearch, 1:200) or donkey anti-rabbit-Rhodamine Red X (#711–295-152, Jackson ImmunoResearch, 1:400) and donkey anti-mouse-Alexa Fluor 488 (Jackson ImmunoResearch, 1:400) for 40 minutes at room temperature. Sections were mounted in Mowiol (#81381, Sigma-Aldrich) containing TOPRO-3 (#T3605, Invitrogen). In secondary only controls, primary antibodies were omitted to control for non-specific binding.

Wheeler M.A., Jaronen M., Covacu R., Zandee S.E., Scalisi G., Rothhammer V., Tjon E.C., Chao C.C., Kenison J.E., Blain M., Rao V.T., Hewson P., Barroso A., Gutiérrez-Vázquez C., Prat A., Antel J.P., Hauser R, & Quintana F.J. (2019). Environmental control of astrocyte pathogenic activities in CNS inflammation. Cell, 176(3), 581-596.e18.

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