Donkey anti chicken alexa fluor 488

Manufactured by Thermo Fisher Scientific

Donkey anti-chicken Alexa Fluor 488 is a secondary antibody used in immunofluorescence applications. It is produced by immunizing donkeys with chicken IgG and conjugating the resulting antibodies with Alexa Fluor 488 dye. This product is designed to detect and visualize chicken primary antibodies in biological samples.

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Lab products found in correlation

Anti fade prolong gold, by Thermo Fisher Scientific (5 mentions) Rabbit anti laminin, by Abcam (4 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Alexa fluor 647 donkey anti goat, by Thermo Fisher Scientific (3 mentions) Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (2 mentions) Alexa fluor 594 donkey anti mouse, by Thermo Fisher Scientific (2 mentions) Alexa fluor 594 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions) Anti cd41 apc, by Thermo Fisher Scientific (2 mentions) Rabbit anti perilipin, by Merck Group (2 mentions) Goat anti c kit, by R&D Systems (2 mentions) Cryostat, by Leica (2 mentions) Sp8 confocal microscope, by Leica (2 mentions) Alexa fluor 647 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions) Lsm780 confocal microscope, by Leica (2 mentions) Mouse anti acetylated α tubulin, by Merck Group (1 mentions) Tcs spe laser scanning confocal system, by Leica (1 mentions) Chick anti gfp, by Abcam (1 mentions) Dm550b, by Leica (1 mentions) Application suite advanced fluorescence 3 0 0 build 8134, by Leica (1 mentions) Mouse anti neun antibody mab377, by Merck Group (1 mentions)

Spelling variants of this product

Alexa Fluor 488 (6806 citations) Alexa 488 (1863 citations) Alexa Fluors (21 citations) Alexa Fluor 488 anti-chicken (10 citations) Alexa 488 anti-chicken (10 citations) Alexa Fluor 488 anti (6 citations) Donkey anti-chicken Alexa 488 (4 citations) Donkey Alexa-Fluor-488) (3 citations) 488 anti-chicken (2 citations) Alexa Fluor 488-conjugated donkey anti-chicken IgG (2 citations)

11 protocols using donkey anti chicken alexa fluor 488

Immunostaining of Mouse Testicular Tissue

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Frozen and paraffin sections of mouse testes were prepared as described (Nagy et al., 2003 ). Dry-down slides of sperm were also used. Samples were fixed in 4% paraformaldehyde for 10 minutes, permeabilized in 0.5% Triton X-100/PBS for 15 minutes and incubated for 1–2 hours in blocking solution (3% BSA and 0.02% Triton X-100 in PBS). Standard procedures were used for immunostaining (Ausubel et al., 2003 ). Primary antibodies used included mouse anti-acetylated-α-tubulin (Sigma, 1:500), chick anti-GFP (Abcam, 1:500), rabbit anti-STK36 (Proteintech, 1:50) and rabbit anti-TEX14 (Abcam, 1:400). Secondary antibodies used included donkey anti-mouse Alexa Fluor 594 (Life Technologies, 1:2000), donkey anti-mouse Alexa Fluor 488 (Life Technologies, 1:2000), donkey anti-rabbit Alexa Fluor 594 (Life Technologies, 1:2000) and donkey anti-chicken Alexa Fluor 488 (Life Technologies, 1:2000).
Confocal images were acquired with a Leica TCS SPE laser scanning confocal system. Confocal stacks were collected using a 0.25 µm-0.5 µm step size along the z-axis. NIH ImageJ was used for image analysis.

Nozawa Y.I., Yao E., Gacayan R., Xu S.M, & Chuang P.T. (2014). Mammalian Fused is essential for sperm head shaping and periaxonemal structure formation during spermatogenesis. Developmental biology, 388(2), 170-180.

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Immunostaining of Bone Tissue Sections

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Freshly dissected bones were fixed in 4% paraformaldehyde overnight followed by 3-day decalcification in 10% EDTA. Bones were sectioned (5μm for thin sections and 50μm for thick sections) using the CryoJane tape-transfer system (Instrumedics, Ann Arbor, MI). Sections were blocked in PBS with 10% horse serum for 1 hour and then stained overnight with chicken-anti-GFP (Aves, Tigard, OR, 1:1000), anti-CD41-APC (eBioscience, eBioMWReg30, 1:200), goat-anti-c-kit (R&D, 1:400), rabbit-anti-perilipin (Sigma, 1:1000) and/or rabbit-anti-laminin (Abcam, Cambridge, MA, 1:400) antibodies. Donkey-anti-goat Alexa Fluor 647, donkey-anti-chicken Alexa Fluor 488 and/or Donkey-anti-goat Alexa Fluor 647 were used as secondary antibodies (Life Technologies, 1:400). Slides were mounted with anti-fade prolong gold (Life Technologies) and images were acquired with a Zeiss LSM780 confocal microscope.

Zhou B.O., Yu H., Yue R., Zhao Z., Rios J.J., Naveiras O, & Morrison S.J. (2017). Bone marrow adipocytes promote the regeneration of stem cells and hematopoiesis by secreting SCF. Nature cell biology, 19(8), 891-903.

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Immunostaining of Bone Tissue Sections

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Validating Neural Implant Placement via GFP Immunostaining

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GFP immunocytochemistry was used to confirm placement of surgical injections and placement of the optical fiber. Animals were anesthetized with Euthasol and perfused transcardially with 4% paraformaldehyde. Brains were dissected, post-fixed overnight in 4% PFA, and cryoprotected in 30% sucrose at 4°C for at least 72 hours. Brains were sectioned at a thickness of 50μm using a sliding microtome and sections were incubated in chicken anti-GFP (1:5000; Aves Lab Inc.) primary antibody for 24 hours at 4°C. The sections were rinsed in 0.1M phosphatebuffer (PB) and incubated with donkey anti-chicken Alexa Fluor 488 (1:500; Life Technology, Carlsbad, CA) antibody for 1 hour at room temperature. Sections were imaged using an epifluorescent microscope (Leica DM550B with Leica Application Suite Advanced Fluorescence 3.0.0 build 8134 software, Leica Microsystems, Wetzlar, Germany). Animals with improper bilateral injection or optical fiber placement were excluded from behavioral data analysis.

Hackett J., Nadkarni V., Singh R.S., Carthy C.L., Antigua S., Hall B.S, & Rajadhyaksha A.M. (2023). Repeat investigation during social preference behavior is suppressed in male mice with prefrontal cortex cacna1c (Cav1.2)-deficiency through the dysregulation of neural dynamics. bioRxiv.

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Immunohistochemical staining of NeuN and GFP

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Mouse anti-NeuN antibody (MAB377) was obtained from Millipore (Billerica, MA). Anti-GFP antibody (Cat #AB290) was purchased from Abcam (Cambridge, UK). Donkey anti-mouse IgG AlexaFluor 564, donkey anti-chicken AlexaFluor 488 antibodies, were purchased from Life Technologies (Grand Island, NY). Ethyl alcohol (190 proof) was purchased from VWR (Radnor, PA). Saccharin (saccharin sodium salt hydrate) was purchased from Sigma Aldrich (St. Louis, MO). Other common reagents were from Sigma Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).

Moffat J.J., Sakhai S.A., Ehinger Y., Phamluong K., & Ron D. (2021). BRAIN-DERIVED NEUROTROPHIC FACTOR IN AN ORBITOFRONTAL CORTICAL-DORSOLATERAL STRIATAL CIRCUIT GATES ALCOHOL CONSUMPTION.

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Multimodal Imaging of Bone Marrow

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Freshly dissected bones were fixed in 4% paraformaldehyde overnight followed by 3-day decalcification in 10% EDTA. Bones were sectioned using the CryoJane tape-transfer system (Instrumedics, St. Louis, MO). Sections were blocked in PBS with 10% horse serum for 1 hr and then stained overnight with goat-anti-Angpt1 (Santa Cruz, Dallas, TX, 1:200), chicken-anti-GFP (Aves, Tigard, OR, 1:1000), anti-CD41-PE (eBioscience, clone eBioMWReg30, 1:200) and/or goat-anti-Osteopontin (R&D, Minneapolis, MN, 1:400) antibodies. Donkey-anti-goat Alexa Fluor 647, donkey-anti-chicken Alexa Fluor 488, and/or Donkey-anti-goat Alexa Fluor 555 were used as secondary antibodies (Invitrogen, Grand Island, NY, 1:400). Slides were mounted with anti-fade prolong gold (Invitrogen) and images were acquired with a LSM780 confocal microscope (Zeiss, San Diego, CA). For thick sections, the specimens were cleared overnight with Benzyl Alcohol/Benzyl Benzoate (1:2) solution (Sigma). 3D reconstruction of bone marrow was achieved by Z stack of tiled images of femoral bone marrow with a Zeiss LSM780 confocal microscope.

Zhou B.O., Ding L, & Morrison S.J. (2015). Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting Angiopoietin-1. eLife, 4, e05521.

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Cryosectioning and Immunostaining Bone and Spleen

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For bone marrow sections, freshly dissected bones were fixed in 4% paraformaldehyde overnight followed by 3 days of decalcification in 10% EDTA dissolved in PBS. Bones were sectioned using the CryoJane tape-transfer system (Instrumedics). For spleen sections, freshly dissected spleens were fixed in 4% paraformaldehyde for 1 hour followed by 1 day incubation in 10% Sucrose in PBS. Frozen spleens were sectioned with a cryostat (Leica). For whole mount imaging, spleens were sectioned into ~2 mm pieces. Spleen sections were blocked in PBS with 10% horse serum for 1 hour and then stained overnight with chicken-anti-GFP (Aves), and/or rabbit-anti-Laminin (Abcam) antibodies. Donkey-anti-chicken Alexa Fluor 488 and/or Donkey-anti-rabbit Alexa Fluor 647 were used as secondary antibodies (Invitrogen). Specimens were mounted with anti-fade prolong gold (Invitrogen) and images were acquired with either a Zeiss LSM780 confocal microscope or a Leica SP8 confocal microscope equipped with a resonant scanner. Three dimensional images were achieved using Bitplane Imaris v7.7.1 software.

Inra C.N., Zhou B.O., Acar M., Murphy M.M., Richardson J., Zhao Z, & Morrison S.J. (2015). A perisinusoidal niche for extramedullary haematopoiesis in the spleen. Nature, 527(7579), 466-471.

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Cryosectioning and Immunostaining Bone and Spleen

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Inra C.N., Zhou B.O., Acar M., Murphy M.M., Richardson J., Zhao Z, & Morrison S.J. (2015). A perisinusoidal niche for extramedullary haematopoiesis in the spleen. Nature, 527(7579), 466-471.

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GFP Immunostaining in Brain Slices

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After electrophysiological recordings, brain slices were fixed in 4% paraformaldehyde overnight. Washing was done with a step-wise protocol using a tris-buffered saline, and 0.3% triton solution. Slices were transferred to the same tris-buffered saline containing donkey serum (5%) for 2 h to block unspecific binding of antibodies. Incubation with a primary anti-GFP antibody (1:1000; polyclonal chicken, Abcam) in 5% donkey serum was done for 72 h at 4°C. Subsequently, slices were rinsed with tris-buffered saline and incubated with donkey-anti-chicken-Alexa Fluor 488 (1:500; Invitrogen) and Alexa Fluor 568 conjugated Streptavidin (1:1000; MoBiTec) for 72 h at 4°C. After the final rinsing, slices were mounted with ProLong Gold Antifade (Invitrogen), and imaged using a Zeiss LSM900 confocal microscope (Oberkochen, Germany). Image analysis and processing was done using the Zeiss ZEN software and ImageJ freeware¹.

Schulz J.M., Kay J.W., Bischofberger J, & Larkum M.E. (2021). GABAB Receptor-Mediated Regulation of Dendro-Somatic Synergy in Layer 5 Pyramidal Neurons. Frontiers in Cellular Neuroscience, 15, 718413.

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Immunohistochemical Analysis of Brain Tissue

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Tissue preparation and immunohistochemistry were done as described (Palop et al., 2011 (link)). After fixation, brains were cryoprotected in 30% sucrose in phosphate-buffered saline (PBS) for 2 days and sectioned with a sliding microtome (Leica Microsystems) into 30-µm sections. Ten stereological subseries sections from each hemibrain were stored in a solution containing 30% glycerol, 30% ethylene glycol, and 40% PBS at −20°C until staining. Primary antibodies used included rabbit anti-GFP (1:3,000; Life Technologies), chicken anti-GFP (1:500; Aves Labs), rabbit anti-Nav1.1 (1:500; Alomone), mouse anti-parvalbumin (1:3,000; Swant), rabbit anti-parvalbumin (1:3,000; Swant), rat anti-somatostatin (1:50; Novus), rabbit anti-neuropeptide Y (1:2000; Immunostar), rabbit anti-Iba-1 (1:1000; Wako), rabbit anti-GFAP (1:3000; Sigma), and biotinylated 82E1 (1:1000; Immuno-Biological Laboratories). Primary antibodies were detected with biotinylated donkey anti-rabbit (1:500; Jackson ImmunoResearch), Alexa Fluor 488 donkey anti-rabbit (1:300; Life Technologies), Alexa Fluor 488 donkey anti-chicken (1:300; Life Technologies), Alexa Fluor 594 donkey anti-rat (1:300; Life Technologies), Alexa Fluor 594 donkey anti-mouse (1:300; Life Technologies), or Alexa Fluor 594 donkey anti-rabbit (1:300; Life Technologies).

Martinez-Losa M., Tracy T.E., Ma K., Verret L., Clemente-Perez A., Khan A.S., Cobos I., Ho K., Gan L., Mucke L., Alvarez-Dolado M, & Palop J.J. (2018). Nav1.1-Overexpressing Interneuron Transplants Restore Brain Rhythms and Cognition in a Mouse Model of Alzheimer’s Disease. Neuron, 98(1), 75-89.e5.

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