Cy2 donkey anti rabbit

Manufactured by Jackson ImmunoResearch

Sourced in Germany

Cy2-donkey anti-rabbit is a secondary antibody conjugated with the fluorescent dye Cy2. It is designed to detect and label rabbit primary antibodies in various immunological applications.

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Lab products found in correlation

Donkey anti rabbit cy3, by Jackson ImmunoResearch (8 mentions) Donkey anti mouse cy3, by Jackson ImmunoResearch (3 mentions) Donkey anti mouse cy2, by Jackson ImmunoResearch (3 mentions) Cy3 donkey anti guinea pig, by Jackson ImmunoResearch (3 mentions) Vectashield, by Vector Laboratories (3 mentions) Tomato lectin fluorescein, by Vector Laboratories (2 mentions) Donkey anti goat cy5, by Jackson ImmunoResearch (2 mentions) Triton x 100, by Thermo Fisher Scientific (2 mentions) Mouse anti neun, by Merck Group (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Mouse α mapk yt, by Merck Group (2 mentions) Goat anti mouse cy3, by Jackson ImmunoResearch (2 mentions) Goat anti rabbit cy3, by Jackson ImmunoResearch (2 mentions) Rabbit anti foxg1, by Abcam (1 mentions) Mouse anti oct3 4, by Santa Cruz Biotechnology (1 mentions) Mouse anti map2, by Abcam (1 mentions) Rabbit anti pax6, by BioLegend (1 mentions) Rabbit anti tbr1, by Abcam (1 mentions) Dapi nuclear stain, by Merck Group (1 mentions) Dmi6000 b, by Adobe (1 mentions)

12 protocols using cy2 donkey anti rabbit

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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Multicolor Immunofluorescence of Vasopressin

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The following primary and secondary antibodies were used: anti-AVP (rabbit, ImmunoStar, 20069, Hudson, WI); antitype IV collagen (goat, SouthernBiotech, 1340-01, Birmingham, AL); anti-SMA (1:67, mouse, Dako, M0851, Santa Clara, CA): donkey antirabbit Cy2 (Jackson ImmunoResearch, 711-225-152, West Grove, PA); donkey antirabbit Cy3 (Jackson ImmunoResearch, 711-165-152); donkey antimouse Cy3 (Jackson ImmunoResearch, 715-165-151); donkey antigoat Cy5 (Jackson ImmunoResearch, 705-175-147); tomato lectin-fluorescein (Vector laboratories, FL-1171, Burlingame, CA).

Yao Y., Taub A.B., LeSauter J, & Silver R. (2021). Identification of the suprachiasmatic nucleus venous portal system in the mammalian brain. Nature Communications, 12, 5643.

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Immunohistochemical Staining of Brain Sections

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Sections (six per animal) were washed in PBS for 30 min and incubated in blocking solution (3% normal donkey serum (NDS), 0.3% Triton X-100 in PBS) for 2 h at room temperature under gentle shaking. Sections were then incubated in primary antibody solution overnight at 4°C under gentle shaking (3% NDS, 0.3% Tween-20 in PBS, with either guinea pig anti-VGLUT1 (1:10,000); guinea pig anti-VGLUT2 (Millipore, #ab2251) 1:10,000; goat anti-PSD95 1:1:1,000; rabbit anti-TH (Millipore, #ab152) 1:10,000). Sections were washed in PBS for 1h, incubated in secondary antibody for 2 h under gentle shaking (donkey anti-rabbit Cy2 1:200; donkey anti-goat Cy2 1:200; donkey anti-guinea pig Cy3 1:200 (Jackson ImmunoResearch). After 1h washing in PBS, slices were briefly incubated with DAPI (1 μg/ml). All sections were mounted onto superfrost plus slides, allowed to dry overnight, dehydrated in alcohol and cleared in Citrisolv, then coverslipped using DPX mounting medium (Electron Microscopy Sciences).

Brancato A., Bregman D., Ahn H.F., Pfau M.L., Menard C., Cannizzaro C., Russo S.J, & Hodes G.E. (2017). Sub-chronic variable stress induces sex-specific effects on glutamatergic synapses in the nucleus accumbens. Neuroscience, 350, 180-189.

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Multimarker Labeling of Vasculature and SCN

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The following primary antibodies were used in the iDISCO protocol: for labeling the SCN, AVP antibody (1:2500, rabbit; ImmunoStar, Hudson, WI); for labeling arteries, smooth muscle actin (SMA) antibody (1:67, mouse; Dako, M0851, Santa Clara, CA); for labeling the entire vasculature, Type IV Collagen-UNLB antibody (1:50, goat; SouthernBiotech, Birmingham, AL). For the sections, the rabbit AVP antibody was used at 1:5000. The following secondary antibodies were used in the iDISCO protocol; for AVP, donkey anti-rabbit Cy2 (1:200; Jackson ImmunoResearch); for SMA, donkey anti-mouse Cy3 (1:200; Jackson ImmunoResearch, West Grove, PA); for collagen, donkey anti-goat Cy5 (1:200; Jackson ImmunoResearch), and for sections, for AVP, donkey anti-rabbit Cy3 (1:200; Jackson ImmunoResearch); for the entire vasculature, tomato lectin-fluorescein (1:100; Vector Labs, Newark, CA).

Bacigalupo J., Johnson E.C., Vergara C, & Lisman J.E. (1991). Light-dependent channels from excised patches of Limulus ventral photoreceptors are opened by cGMP.. Proceedings of the National Academy of Sciences of the United States of America, 88(18), 7938-7942.

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Quantifying pMeCP2 and Fos in Brain

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Brains were cut to 40µm coronal sections on a freezing microtome, and brain regions (prelimbic and infralimbic regions of the medial prefrontal cortex, nucleus accumbens, and basolateral amygdala) were identified by anatomical landmarks. Sections were blocked in 10% normal goat serum with 0.3% Triton X-100 in PBS and were incubated overnight in primary antibodies, which included rabbit anti-phospho-Ser421 MeCP2 (1:15,000; (Deng et al. 2010 (link))) and rabbit anti-Fos (1:10,000; Ab-5; Millipore). Because both antibodies were raised in rabbit, sections could not be co-stained for both pMeCP2-S421 and Fos; thus we used adjacent sections from each animal for these analyses. Sections were visualized by application of donkey anti-rabbit Cy3 or donkey anti-rabbit Cy2 (1:500; Jackson ImmunoResearch) secondary antibodies. Hoechst dye was added to visualize nuclei and the sections were mounted in Vectashield (Vector Laboratories). To minimize bleaching, sections were stored at all stages in dark conditions and were exposed for the minimum duration of time to bright field illumination.

Zannas A.S., Kim J.H, & West A.E. (2017). Regulation and function of MeCP2 Ser421 phosphorylation in U50488-induced conditioned place aversion in mice. Psychopharmacology, 234(6), 913-923.

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Immunohistochemical Characterization of Opsin Expression

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After the electrophysiological experiments, mice were transcardially perfused with 4% paraformaldehyde (PFA) under deep isoflurane anesthesia to characterize the opsin expression. Brains were removed, fixed overnight in 4% PFA, and transferred to 30% sucrose solution. Coronal brain sections of 70 μm thickness were prepared using a vibratome (Leica, Wetzlar, Germany). For permeabilization, slices were incubated with 0.1% Triton X-100 and 5% normal donkey serum (Invitrogen, Life Technologies, Carlsbad, CA) in phosphate buffer solution for 90 min. Slices were incubated with goat anti-PV (1:200, Swant, Marly, Switzerland) or rabbit anti-CamKII (1:200, Epitomics, Burlingame, CA) at 4 °C overnight. On the next day, slices were incubated with the secondary antibodies Cy-2 donkey anti-goat (1:200, Jackson Immuno Research, West Grove, PA), or Cy-2 donkey anti-rabbit (1:200, Jackson Immuno Research, West Grove, PA), and a fluorescent Nissl stain (red 615 nm, Neurotrace, Molecular Probes, Life Technologies, Carlsbad, CA). Slices were mounted using antiquenching Vectashield (Vector Laboratories, Burlingame, CA).

Yang J.W., Prouvot P.H., Reyes-Puerta V., Stüttgen M.C., Stroh A, & Luhmann H.J. (2017). Optogenetic Modulation of a Minor Fraction of Parvalbumin-Positive Interneurons Specifically Affects Spatiotemporal Dynamics of Spontaneous and Sensory-Evoked Activity in Mouse Somatosensory Cortex in Vivo. Cerebral Cortex (New York, NY), 27(12), 5784-5803.

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Immunocytochemistry of Induced Neurons

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Coverslips (Electron Microscopy Sciences #7229102) were coated with poly-L-ornithine, laminin, and Matrigel as descrbied above for routine iN cultures. On DIV21, iNs were washed once with PBS, then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Following fixation, iNs were washed with PBS for 15 minutes at room temperature then incubated in blocking buffer (2% donkey serum with 0.1% Triton X-100) for 1 h shaking at room temperature. iNs were incubated in primary antibodies diluted in blocking buffer overnight at 4 °C. Following incubation iNs were washed with PBS 3 times for 10–15 min and incubated in secondary antibodies for 1 h at room temperature. Secondaries included Cy2 Donkey anti-rabbit, Cy2 Donkey anti-mouse, Cy3-Donkey anti-mouse, Cy3-Donkey anti-rabbit, Cy5-Donkey anti-chicken 1:2000, (Jackson Immunoresearch). iNs were then washed with PBS 3 times. DAPI (1:1000) staining was performed during the second wash with PBS. Coverslips were mounted and imaged using Zeiss LSM710 confocal microscopy, 40× oil objective and acquired using Zen black software.

White A.J., Clark K.A., Alexander K.D., Ramalingam N., Young-Pearse T.L., Dettmer U., Selkoe D.J, & Ho G.P. (2024). A stem cell-based assay platform demonstrates alpha-synuclein dependent synaptic dysfunction in patient-derived cortical neurons. NPJ Parkinson's Disease, 10, 107.

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Immunostaining of Muscle Tissue

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Muscle cross‐section was fixed with 4% PFA at room temperature for 10 minutes, washed (2 times for 3 minutes each) with PBS‐T (PBS with 0.1% Tween 20) and incubated for 1 hour with blocking solution (1% bovine serum albumin in PBS‐T). The membranes were then incubated overnight at 4ºC with primary antibody rabbit anti‐eMHC (1:500, Origene cat#TA349138), rabbit anti‐GFP (1:250, Invitrogen cat#A10260), rabbit anti‐Ki67 (1:200, Cell Signaling cat#9129), rabbit anti‐Pax7 (1:50, Abcam cat#ab34360) or rabbit anti‐MyoD (1:50, Santa Cruz cat# sc‐304). After washing with PBS‐T 0.1% (3 times for 5 minutes each), secondary antibody (1:250 Cy3 Donkey Anti‐Rabbit or Cy2 Donkey Anti‐Rabbit, both Jackson ImmunoResearch) was added in blocking solution for 1 hour in the dark, followed by further washing three times for 5 minutes in PBS‐T. Finally, slides were mounted with coverslips with Vectashield for fluorescence with 4′,6‐diamidino‐2‐phenylindole (DAPI) (cat# H‐1200, Vector Labs). Digital capture of the stained sections was performed with a fluorescence microscope Axio Scope.A1 (Carl Zeiss Microscopy GmbH, Göttingen, Germany).

Silva W.J., Graça F.A., Cruz A., Silvestre J.G., Labeit S., Miyabara E.H., Yan C.Y., Wang D.Z, & Moriscot A.S. (2019). miR‐29c improves skeletal muscle mass and function throughout myocyte proliferation and differentiation and by repressing atrophy‐related genes. Acta Physiologica (Oxford, England), 226(4), e13278.

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Immunostaining of DRG Neuron Development

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DRG neurons of different time of growth (from 3 to 12 DIV) were fixed for 20 min with 4% paraformaldehyde (Sigma) rinsed in PBS (0.01 M, pH 7.4) at room temperature, followed by washes with PBS, blocking in 5% BSA (Sigma) with 0.5% of triton (Sigma), and incubation with primary antibodies overnight at 4℃ in 1% BSA with 0.05% of triton. Following day, the primary antibodies were washed out by 1 × PBS and followed by incubation with Cy2, Cy3-conjungated secondary antibodies (Jackson ImmunoResearch) for 2 h at T_room in dark.
Primary antibodies: goat anti-Nav1.8 (1:50, Santa Cruz),^{22 (link)} rabbit anti-TRPV1 (1:50, Santa Cruz),^{23 (link)–25 (link, link)} rabbit anti-TRPM8 (1:50, Santa Cruz), rat anti-CD77 (1:10, Abcam),^{26 (link)} mouse anti-NeuN (1:2000, Millipore),^{27 (link)} and rabbit anti-Tuj1 (β3 Tubulin, 1:50, Santa Cruz).^{28 (link)}Secondary antibodies: Cy2-donkey anti-rabbit, Cy2-donkey anti-mouse, Cy3-donkey anti-rat, and Cy3-donkey anti-rabbit (1:200, all from Jackson ImmunoResearch). DAPI (dilactate; 300 nM, Invitrogen) was used for counterstaining.

Lakomá J., Rimondini R., Ferrer Montiel A., Donadio V., Liguori R, & Caprini M. (2016). Increased expression of Trpv1 in peripheral terminals mediates thermal nociception in Fabry disease mouse model. Molecular Pain, 12, 1744806916663729.

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Antibody panel for LRP1, PID1, GLUT4 analysis

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Antibodies used in the study were: sheep polyclonal anti-LRP1 (made in-house, IF 1:250; IP 1:200 [33 (link)]), rabbit monoclonal anti-LRP1 (abgent, AJ1448a; WB 1:10000), rabbit polyclonal anti-PID1 (Sigma-Aldrich, HPA36103; WB 1:200), rabbit polyclonal anti-GLUT4 (kindly provided by A. Schürmann, DIfE, Potsdam-Rehbrücke, Germany; WB 1:1000, IF 1:500), rabbit monoclonal anti-IRAP (Cell Signaling 6918, IF 1:250), mouse monoclonal anti-GLUT1 (Abcam, ab40084, IF 1:100), rabbit polyclonal anti-AS160 (Millipore 07–741, WB 1:500), mouse monoclonal anti-GST (Santa Cruz sc-138, WB 1:500), rabbit polyclonal anti-AKT (Cell Signaling, 9272, WB 1:1000) and rabbit polyclonal anti-p-AKT (Ser473) (Cell Signaling, 9271, WB 1:1000).
All following secondary antibodies were purchased from Jackson Immuno Research: goat anti-rabbit HRP (#111-035-144, WB 1:5000); goat anti-mouse HRP (#115-035-003, WB 1:5000); Cy2-donkey-anti-sheep (#713-225-147, IF 1:250), Cy2-donkey-anti-sheep (#713-166-147, IF 1:500), Cy2-donkey-anti-rabbit (#711-225-152, IF 1:250), Cy3-donkey-anti-rabbit (#711-165-152; IF 1:500), Alexa488-donkey-anti-mouse (#715-486-150, IF 1:250).

Fischer A.W., Albers K., Schlein C., Sass F., Krott L.M., Schmale H., Gordts P.L., Scheja L, & Heeren J. (2019). PID1 regulates insulin-dependent glucose uptake by controlling intracellular sorting of GLUT4-storage vesicles. Biochimica et biophysica acta. Molecular basis of disease, 1865(6), 1592-1603.

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