Donkey anti chicken cy3

Manufactured by Jackson ImmunoResearch

Sourced in Germany

Donkey anti-Chicken Cy3 is a secondary antibody conjugated with the fluorescent dye Cy3. It is designed to detect and visualize chicken primary antibodies in various immunological techniques, such as immunofluorescence, flow cytometry, and Western blotting.

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Close spelling variants of this product

Cy3 donkey anti-chicken IgY Anti-chicken Cy3

4 protocols using donkey anti chicken cy3

Immunofluorescence Staining of Cultured Cells

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Cultured cells were fixed with 4% PFA for 15 minutes at room temperature (RT) and block-permeablised using PBST-10% Normal Donkey Serum (NDS) for 1 hour at RT. Primary and secondary antibodies were incubated in 3%NDS overnight (O/N) at 4 °C and 1 hour at RT respectively at the following dilutions; mouse anti-CNPAse, (1:2000; Chemicon, Germany), rabbit anti-GFAP, goat anti-GFAP, mouse anti-βIII-tubulin, rabbit anti-βIII-tubulin (all at 1:300; Sigma-Aldrich), rabbit anti-Pax6 (1:200; Chemicon, Germany), donkey anti-sheep Alexafluor555, donkey anti-rabbit Alexafluor488/555/647 and donkey anti-mouse Alexafluor488/555/647 (all 1:1000; Invitrogen), donkey anti-chickenCy3 (Jackson Laboratories, Bar Harbour, ME). Cells were counterstained with DAPI and mounted with Slow-fade mounting media (Invitrogen). Non-specific staining was controlled by using secondary-only controls. Fluorescence was viewed using the Axioplan2 microscope (Carl Zeiss, Jena, Germany) fitted with an HBO 100 lamp (Carl Zeiss, Jena, Germany). Images were captured using an Axiocam Mrm camera and Axio Vs40 v4.5.0.0 software (Axiovision, Carl Zeiss, Jena, Germany).

Bridges C.R., Tan M.C., Premarathne S., Nanayakkara D., Bellette B., Zencak D., Domingo D., Gecz J., Murtaza M., Jolly L.A, & Wood S.A. (2017). USP9X deubiquitylating enzyme maintains RAPTOR protein levels, mTORC1 signalling and proliferation in neural progenitors. Scientific Reports, 7, 391.

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Drosophila Larval Tissue Staining

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Drosophila larva transitions from larval stage L1 to larval stage L3 by intermittent molting. Prior to pupariation, L3 larvae stop feeding and migrate to a pupariation site on the side of a vial. Wandering L3 larvae were bisected and inverted in PBS, and all tissues were fixed in 4% formaldehyde/PBS for 30 min, permeabelized in 0.2% Triton X-100/PBS for 4 hours, and blocked in 3% BSA/PBS for 1 hour. All antibody stainings were performed in 3% BSA/PBS, incubation of primary and secondary antibodies were O/N. PBS was used for all rinses and washes (3× each for primary and secondary antibody incubation steps). Antibodies used were as follows: Chicken anti-RFP 1:2,000 (Rockland, 600-901-379); Goat anti-GFP 1:3,000 (Rockland, 600-101-215); Donkey anti-Goat Alexa Fluor 488 (Life Technologies, A11055); Donkey anti-Chicken Cy3 (Jackson ImmunoResearch, 703-165-155); and Donkey anti-Mouse Alexa Fluor 594 (Life Technologies, A21203). All secondary antibodies were used at 1:500. Tissues were dissected off the cuticle and were mounted in SlowFade Gold mounting medium with DAPI (Life Technologies, S36938). See Kockel et al. (2016) (link) for a detailed protocol.

Kim E.S., Rajan A., Chang K., Govindarajan S., Gulick C., English E., Rodriguez B., Bloomfield O., Nakada S., Beard C., O’Connor S., Mastroianni S., Downey E., Feigenbaum M., Tolentino C., Pace A., Khan M., Moon S., DiPrima J., Syed A., Lin F., Abukhadra Y., Bacon I., Beckerle J., Cho S., Donkor N.E., Garberg L., Harrington A., Hoang M., Lawani N., Noori A., Park E., Parsons E., Oravitan P., Chen M., Molina C., Richmond C., Reddi A., Huang J., Shugrue C., Coviello R., Unver S., Indelicarto M., Islamovic E., McIlroy R., Yang A., Hamad M., Griffin E., Ahmed Z., Alla A., Fitzgerald P., Choi A., Das T., Cheng Y., Yu J., Roderiques T., Lee E., Liu L., Harper J., Wang J., Suhr C., Tan M., Luque J., Tam A.R., Chen E., Triff M., Zimmermann L., Zhang E., Wood J., Clark K., Kpodonu N., Dey A., Ecker A., Chuang M., López R.K., Sun H., Wei Z., Stone H., Chi C.Y., Silvestri A., Orloff P., Nedumaran N., Zou A., Ünver L., Page O., Kim M., Chan T.Y., Tulloch A., Hernandez A., Pillai A., Chen C., Chowdhury N., Huang L., Mudide A., Paik G., Wingate A., Quinn L., Conybere C., Baumgardt L.L., Buckley R., Kolberg Z., Pattison R., Shazli A.A., Ganske P., Sfragara L., Strub A., Collier B., Tamana H., Ravindran D., Howden J., Stewart M., Shimizu S., Braniff J., Fong M., Gutman L., Irvine D., Malholtra S., Medina J., Park J., Yin A., Abromavage H., Barrett B., Chen J., Cho R., Dilatush M., Gaw G., Gu C., Huang J., Kilby H., Markel E., McClure K., Phillips W., Polaski B., Roselli A., Saint-Cyr S., Shin E., Tatum K., Tumpunyawat T., Wetherill L., Ptaszynska S., Zeleznik M., Pesendorfer A., Nolan A., Tao J., Sammeta D., Nicholson L., Dinh G.V., Foltz M., Vo A., Ross M., Tokarski A., Hariharan S., Wang E., Baziuk M., Tay A., Wong Y.H., Floyd J., Cui A., Pierre K., Coppisetti N., Kutam M., Khurjekar D., Gadzi A., Gubbay B., Pedretti S., Belovich S., Yeung T., Fey M., Shaffer L., Li A., Beritela G., Huyghue K., Foster G., Durso-Finley G., Thierfelder Q., Kiernan H., Lenkowsky A., Thomas T., Cheng N., Chao O., L’Etoile-Goga P., King A., McKinley P., Read N., Milberg D., Lin L., Wong M., Gilman I., Brown S., Chen L., Kosai J., Verbinsky M., Belshaw-Hood A., Lee H., Zhou C., Lobo M., Tse A., Tran K., Lewis K., Sonawane P., Ngo J., Zuzga S., Chow L., Huynh V., Yang W., Lim S., Stites B., Chang S., Cruz-Balleza R., Pelta M., Kujawski S., Yuan C., Standen-Bloom E., Witt O., Anders K., Duane A., Huynh N., Lester B., Fung-Lee S., Fung M., Situ M., Canigiula P., Dijkgraaf M., Romero W., Baula S.K., Wong K., Xu I., Martinez B., Nuygen R., Norris L., Nijensohn N., Altman N., Maajid E., Burkhardt O., Chanda J., Doscher C., Gopal A., Good A., Good J., Herrera N., Lanting L., Liem S., Marks A., McLaughlin E., Lee A., Mohr C., Patton E., Pyarali N., Oczon C., Richards D., Good N., Goss S., Khan A., Madonia R., Mitchell V., Sun N., Vranka T., Garcia D., Arroyo F., Morales E., Camey S., Cano G., Bernabe A., Arroyo J., Lopez Y., Gonzalez E., Zumba B., Garcia J., Vargas E., Trinidad A., Candelaria N., Valdez V., Campuzano F., Pereznegron E., Medrano J., Gutierrez J., Gutierrez E., Abrego E.T., Gutierrez D., Ortiz C., Barnes A., Arms E., Mitchell L., Balanzá C., Bradford J., Detroy H., Ferguson D., Guillermo E., Manapragada A., Nanula D., Serna B., Singh K., Sramaty E., Wells B., Wiggins M., Dowling M., Schmadeke G., Cafferky S., Good S., Reese M., Fleig M., Gannett A., Cain C., Lee M., Oberto P., Rinehart J., Pan E., Mathis S.A., Joiner J., Barr L., Evans C.J., Baena-Lopez A., Beatty A., Collette J., Smullen R., Suttie J., Chisholm T., Rotondo C., Lewis G., Turner V., Stark L., Fox E., Amirapu A., Park S., Lantz N., Rankin A.E., Kim S.K, & Kockel L. (2023). Generation of LexA enhancer-trap lines in Drosophila by an international scholastic network. G3: Genes|Genomes|Genetics, 13(9), jkad124.

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Multicolor Immunofluorescence Staining

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All tissues were fixed in 4% formaldehyde/PBS for 30 min, permeabilized in 0.2% Triton X-100/PBS for 4 hr, and blocked in 3% BSA/PBS for 1 hr. All antibody stainings were performed in 3% BSA/PBS, incubation of primary and secondary antibodies were overnight at 4°. PBS was used for all rinses and washes (3× each for primary and secondary antibody incubation steps). Antibodies used: Chicken anti-RFP 1:2000 (Rockland, 600-901-379). Goat anti-GFP 1:3000 (Rockland 600-101-215). Mouse anti-Tubulin 1:5000 (Sigma T5168). Donkey anti-Goat Alexa488 (Life Technologies, A11055). Donkey anti-Chicken Cy3 (Jackson ImmunoResearch 703-165-155). Donkey anti-Mouse Alexa594 (Life Technologies A21203). All secondary antibodies were used at 1:500. All samples were mounted in SlowFade Gold mounting medium with DAPI (Life Technologies, S36938).

Kockel L., Huq L.M., Ayyar A., Herold E., MacAlpine E., Logan M., Savvides C., Kim G.E., Chen J., Clark T., Duong T., Fazel-Rezai V., Havey D., Han S., Jagadeesan R., Kim E.S., Lee D., Lombardo K., Piyale I., Shi H., Stahr L., Tung D., Tayvah U., Wang F., Wang J.H., Xiao S., Topper S.M., Park S., Rotondo C., Rankin A.E., Chisholm T.W, & Kim S.K. (2016). A Drosophila LexA Enhancer-Trap Resource for Developmental Biology and Neuroendocrine Research. G3: Genes|Genomes|Genetics, 6(10), 3017-3026.

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Primary DRG Neuron Culture Protocol

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Primary DRG neuron cultures were fixed in 4% paraformaldehyde for 15 minutes at RT, followed by 2 rinses in PBS. Cells were permeabilized in 0.3% Triton-X100 (Sigma, T8787) in PBS for 5 minutes at RT. Cells were incubated in blocking buffer, 2% FBS (Atlanta Biologicals), 2% heat shock fraction V BSA (Fisher Scientific, BP1600), 0.3% Triton-x-100 in PBS for 1 hour. Cells were incubated with anti-Neurofilament heavy chain (1:100; Aves Lab, NFH) in blocking buffer overnight at 4°C and rinsed 3x in 0.3% triton-x-100 in PBS, 5 minutes each. Donkey anti-chicken Cy3 (1:200, Jackson, 703-165-155) in blocking buffer was added for 45 minutes at room temperature. Cells were rinsed in PBS for 5 minutes. Hoechst 33342 (1:50,000 in PBS; Invitrogen, H3570) was added for 10 minutes at RT, followed by 2 rinses in PBS. Cells were imaged on a Zeiss Axio Observer Z1 fitted with a Zeiss Axiocam 503 mono camera using the EC PlnN 10x objective. Single plane, tile scans were randomly acquired for each well. For quantification of neurite outgrowth, NFH stained cells with neurites ≥ 30 μm were included in the analyses from randomly acquired tile scans using WIS-Neuromath (Rishal et al. 2013 (link)).

Schmitd L.B., Hafner H., Ward A., Asghari Adib E., Biscola N.P., Kohen R., Patel M., Williamson R.E., Desai E., Bennett J., Saxman G., Athaiya M., Wilborn D., Shumpert J., Zhao X.F., Kawaguchi R., Geschwind D.H., Hoke A., Shrager P., Collins C.A., Havton L.A., Kalinski A.L, & Giger R.J. (2024). Sarm1 is not necessary for activation of neuron-intrinsic growth programs yet required for the Schwann cell repair response and peripheral nerve regeneration. bioRxiv.

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