Cy3 conjugated donkey anti rabbit

Manufactured by Jackson ImmunoResearch

Sourced in United States, United Kingdom

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

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41 protocols using cy3 conjugated donkey anti rabbit

Multilineage Differentiation Assay in Mouse Embryos

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All animal experiments were performed in accordance with Institutional Animal Care and Use Committee of IFOM (project #110/11) approved by the Italian Ministry of Health. Animals were kept on a 12/12-h light/dark cycle (lights on at 07:00 h) with free access to food and water. All animal handlings (sacrifice, etc.) were in accordance with the guidelines established by EU (directive 2010/63/EU). Mice were sacrificed by carbon dioxide euthanasia.
E3.5 embryos were flushed according to standard procedures [36 (link)] and cultured for 24h in M16 (Sigma) medium at 37°C and 0.5% CO2. E4.5 embryos were then fixed in PFA 4%, permeabilized in PBS/0.25% Triton X-100, blocked in PBS/0.05% Triton X-100/3% BSA and incubated with primary antibodies: mouse anti-Cdx2 (Biocare Medical CM226; 1:100), rabbit anti-Nanog (Cosmo Bio RCAB0002P-F, 1:50), goat anti-Gata4 (Santa Cruz sc-1237; 1:100), rabbit anti-cleaved Caspase-3 (Cell Signaling 9661; 1:200), followed by secondary antibodies: donkey anti-mouse A488-conjugated (Invitrogen; 1/100), donkey anti-rabbit CY3-conjugated (Jackson Labs; 1/400), donkey anti-goat CY5-conjugated (Jackson Labs; 1/400), and DAPI (0.5μg/mL) counterstaining. The images were acquired with a TCS SP2 AOBS confocal microscope (Leica Microsystem) and processed with ImageJ software.

Laurent A., Calabrese M., Warnatz H.J., Yaspo M.L., Tkachuk V., Torres M., Blasi F, & Penkov D. (2015). ChIP-Seq and RNA-Seq Analyses Identify Components of the Wnt and Fgf Signaling Pathways as Prep1 Target Genes in Mouse Embryonic Stem Cells. PLoS ONE, 10(4), e0122518.

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Immunohistochemical Analysis of KCC2

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Detection of KCC2 was done on 30 μm coronal cryosections from fixed brains (P6 mice, fixed with 4% PFA). Blocking and staining was performed in 3% BSA, 0.2% saponin, 10% goat serum in PBS mounted with Vectashield hard set mounting medium. The following antibodies were used: rabbit anti-KCC2 antibody (1:1,000; Millipore), chicken anti-GFP (1:1,000; Abcam), donkey anti-rabbit Cy3-conjugated (1:1,000; Jackson Immuno Research) and donkey anti-chicken Alexa 488-conjugated (1:1,000; Jackson Immuno Research).

Puskarjov M., Seja P., Heron S.E., Williams T.C., Ahmad F., Iona X., Oliver K.L., Grinton B.E., Vutskits L., Scheffer I.E., Petrou S., Blaesse P., Dibbens L.M., Berkovic S.F, & Kaila K. (2014). A variant of KCC2 from patients with febrile seizures impairs neuronal Cl− extrusion and dendritic spine formation. EMBO Reports, 15(6), 723-729.

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Immunohistochemical Analysis of Neuronal Markers

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Following completion of experiments, rats were anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with 0.01 M PBS (phosphate-buffered saline) followed by 10% buffered formalin solution (HT501320, Sigma Aldrich). Brains were removed and stored in formalin with 20% sucrose. All brains were sectioned at 30 μm on a freezing stage microtome (SM2010R, Leica Biosystems). Sections were collected and processed to label for GFP (as an indicator of GCaMP6f or dLight1.2 expression) and/or TH via immunohistochemistry. Antibodies were incubated at 4 °C (washes and other steps at room temperature). Tissues were permeabilized in 0.3% Triton-X 100 for 30 min and blocked in 2% normal donkey serum for 30 min. Sections were incubated in rabbit anti-TH (AB152, Sigma Aldrich) and/or chicken anti-GFP (AB13907, Abcam) antibodies overnight (∼18 h). After KPBS (potassium phosphate-buffered saline) washes (eight changes, 10 min each), secondary antibody (Cy3 conjugated donkey anti-rabbit and AF488 conjugated donkey anti-chicken; Jackson Immunoresearch) was applied and sections were incubated overnight. Sections were then mounted onto glass slides, air dried, and coverslipped with 50% glycerol in KPBS mountant. Only data from subjects with GFP and mCherry expression and correct fiber placement were included in statistical analyses.

Hsu T.M., Bazzino P., Hurh S.J., Konanur V.R., Roitman J.D, & Roitman M.F. (2020). Thirst recruits phasic dopamine signaling through subfornical organ neurons. Proceedings of the National Academy of Sciences of the United States of America, 117(48), 30744-30754.

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Immunofluorescence and Immunohistochemical Staining

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Immunofluorescence stainings of 4% paraformaldehyde (PFA)-fixed cultured cells were performed in 24 well plates. Immunohistochemical stainings of brain tissues were performed using free-floating sections. Briefly, anesthetized mice were intracardially perfused with 4% PFA in PBS. The brains were removed, fixed overnight in 4% PFA and cut in a Leica VT 1000 S vibratome in 50 μm slices. The slices were permeabilized and blocked using 5% BSA, 0.1% Triton PBS solution for 2 h and were next incubated with the primary antibody using 3% BSA, 0.1% Triton solution overnight. The next day the sections were washed with PBS 3 times and were incubated with the secondary antibody for 4 h in 3% BSA, 0.1% Triton solution. Sections were next washed 3 times with PBS and incubated with DAPI solution for 10 min, washed once again and mounted. Antibodies used were: mouse anti-BrdU (BD Biosciences, cat# 347580), rabbit anti-ACBP (Santa Cruz cat# sc30190), chicken anti-EGFP (Abcam, cat# ab13970), rabbit anti-DsRed (Clontech Living Colors, cat# 632496), mouse anti-ITGB1 (Abcam, cat# ab24693), Alexa 647 conjugated donkey anti-mouse and anti-rabbit (Thermo Fisher, cat# A21447, cat# A31573), Alexa 488 conjugated donkey anti-chicken (Jackson ImmunoResearch Laboratories cat# 703-545-155) and Cy3 conjugated donkey anti-rabbit (Jackson ImmunoResearch Laboratories cat# 711-165-152).

Duman C., Di Marco B., Nevedomskaya E., Ulug B., Lesche R., Christian S, & Alfonso J. (2023). Targeting fatty acid oxidation via Acyl-CoA binding protein hinders glioblastoma invasion. Cell Death & Disease, 14(4), 296.

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Confocal Microscopy of Retrovirally Labeled Neurons

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For confocal microscopy, rats were perfused with ice-cold saline and 4% paraformaldehyde 3 or 6 weeks after retroviral injections. Brains were processed for immunohistochemistry as previously described [11 (link)]. The following primary and secondary antibodies were used: rabbit polyclonal anti-Iba1 (1:1000, Wako, Japan), chicken polyclonal anti-GFP (1:5000, Abcam, UK), mouse monoclonal anti-gephyrin (1:500, Synaptic Systems, Germany), Cy3-conjugated donkey anti-rabbit (1:200, Jackson Immunoresearch, UK), Cy3-conjugated goat anti-mouse (1:200, Jackson Immunoresearch), biotinylated goat anti-chicken (Vector laboratories), Cy5-conjugated goat anti-rabbit (1:200, Jackson Immunoresearch), Alexa-488 goat anti-chicken (1:200, Invitrogen, Sweden), and Alexa-488 streptavidin (1:200, Invitrogen).

Chugh D, & Ekdahl C.T. (2016). Interactions Between Microglia and Newly Formed Hippocampal Neurons in Physiological and Seizure-Induced Inflammatory Environment. Brain Plasticity, 1(2), 215-221.

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Immunoblotting and Immunofluorescence Analyses

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The following antibodies and probes were used where indicated. ATRX (Cell Signaling, D1N2E), ATRX (Santa Cruz H-300), DAXX (Cell Signaling 25C12), FLAG-M2 (Sigma F1804), GAPDH (Santa Cruz, 0411), Histone H3.3 (Abcam, EPR17899), Histone H4 (Active Motif, 39269), PML (Santa Cruz H-238), PML (Santa Cruz PG-M3), α-Tubulin (Cell Signaling, 11H10), Purified Rabbit IgG (Bethyl Laboratories P120-101), Alexa Fluor 488 conjugated Donkey Anti-Rabbit IgG (Jackson ImmunoResearch), Cy3 conjugated Donkey anti-Rabbit (Jackson ImmunoResearch), Peroxidase conjugated Goat Anti-Mouse IgG (Jackson ImmunoResearch), Peroxidase conjugated Goat Anti-Rabbit IgG (Jackson ImmunoResearch). PNA Telomere probe (TelC-Cy3, PNA Bio Inc.) Flag-DAXX/pRK5 was a gift from Xiaolu Yang (Addgene plasmid #27974).

Mason-Osann E., Dai A., Floro J., Lock Y.J., Reiss M., Gali H., Matschulat A., Labadorf A, & Flynn R.L. (2018). Identification of a novel gene fusion in ALT positive osteosarcoma. Oncotarget, 9(67), 32868-32880.

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Immunofluorescence Labeling of Mouse Spinal Cord

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The procedures were described previously^{37 (link),62}. Deeply anesthetized mice (ketamine, 90 mg/kg and Xylazine, 10 mg/kg) were perfused transcardially with 0.01 M PBS (PH 7.4) and paraformaldehyde (PFA) (4% in PBS). Spinal cord and brain were removed and post-fixed in 4% PFA for 2–4 h. The tissues were then cryoprotected in 20% sucrose overnight at 4 °C. Free-floating frozen sections were incubated with 2% donkey serum and 0.3% Triton X-100 for 1 h at room temperature followed by incubation with primary antibodies overnight at 4 °C. The sections were then washed and incubated with secondary antibodies for 2 h at room temperature. The following primary antibodies were used: chicken anti-GFP (1:500, Aves Labs, GFP-1020), guinea-pig anti-NK1R (1:500, AB15810, EMD Millipore), rabbit anti-FG (1:5000, AB153, Millipore). The following secondary antibodies were used: Alexa-Fluor 488 conjugated donkey anti-chicken (1:1000, Jackson ImmunoResearch, 703–545–155), Cy3-conjugated donkey anti-rabbit (1:1000, Jackson ImmunoResearch, 711–165–152) and Cy5-conjugated donkey anti-guinea pig 1(:1000, Jackson ImmunoResearch, 703–175–148). Fluorescent Images were taken using a Nikon C2+ confocal microscope system (Nikon Instruments, Inc.).

Bardoni R., Shen K.F., Li H., Jeffry J., Barry D.M., Comitato A., Li Y.Q, & Chen Z.F. (2019). Pain Inhibits GRPR Neurons via GABAergic Signaling in the Spinal Cord. Scientific Reports, 9, 15804.

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Immunofluorescence Analysis of Pancreatic Tissues

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Deparaffinized pancreatic sections (5 µm) were blocked for 30 min with blocking solution (20% Fetal Bovine Serum + 2% Roche Blocking Reagent). Sections were incubated overnight with primary antibodies and 1–2 h with secondary antibodies at room temperature with the following primary antibodies: guinea pig anti-human insulin (1∶1000; Linco Research), rabbit anti-glucagon (1∶1000, Linco Research), rat anti-mouse CD31 (1∶400; BD Biosciences). Secondary antibodies FITC-conjugated donkey anti-guinea pig, CY3-conjugated donkey anti-rabbit, AMCA-conjugated donkey anti-guinea pig, and CY3-conjugated goat anti-rat (Jackson ImmunoResearch Laboratories) were used at concentrations recommended by the manufacturer. The nuclei were stained with DAPI (Invitrogen, Molecular Probes). Images were obtained with either Nikon Eclipse E400 microscope or Tissue Genostics Tissue/Cell High Throughput Imaging and Analysis System at Northwestern University Cell Imaging Facility.

Kilic G., Alvarez-Mercado A.I., Zarrouki B., Opland D., Liew C.W., Alonso L.C., Myers MG J.r., Jonas J.C., Poitout V., Kulkarni R.N, & Mauvais-Jarvis F. (2014). The Islet Estrogen Receptor-α Is Induced by Hyperglycemia and Protects Against Oxidative Stress-Induced Insulin-Deficient Diabetes. PLoS ONE, 9(2), e87941.

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Visualizing Amph and Dlg Localization

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Larvae were incubated overnight with rabbit anti-Amph antibody (G. Boulianne) and mouse anti-Dlg antibody (DHSB) at 4°C. The samples were incubated for 2 h with Cy3-conjugated donkey anti-rabbit (Jackson Immuno Research Labs) and Alexa Fluor 488–conjugated goat anti-mouse (Invitrogen) antibody to visualize dAmph and Dlg, respectively (Supplemental Table S2).

Safi F., Shteiman-Kotler A., Zhong Y., Iliadi K.G., Boulianne G.L, & Rotin D. (2016). Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation. Molecular Biology of the Cell, 27(6), 907-918.

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Immunofluorescent Detection of p-Smad1/5/8

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Cell spheres were fixed with 4% paraformaldehide (PFA), washed three times with 0.1% phosphate buffer (PB) and incubated in 0.1% PB containing 10% fetal bovine serum and 0.2% Triton X-100 (blocking buffer) for 1 h. Coverslips were incubated with rabbit polyclonal anti– p-Smad1, 5, 8 primary antibody overnight at 4°C. After three washes in 0.1% PB, immunoreactivity was detected with Cy3-conjugated donkey-anti-rabbit (1:1500; Jackson). Cells were counterstained with 4–6-diamidino-2-phenylindole (DAPI), washed in PB, and mounted. Immunofluorescent samples were analyzed and photographed in a Leica Spectral SP5 confocal microscope.

González-Gómez P., Crecente-Campo J., Zahonero C., de la Fuente M., Hernández-Laín A., Mira H., Sánchez-Gómez P, & Garcia-Fuentes M. (2015). Controlled release microspheres loaded with BMP7 suppress primary tumors from human glioblastoma. Oncotarget, 6(13), 10950-10963.

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