Cy5 conjugated donkey anti mouse igg

Manufactured by Jackson ImmunoResearch

Sourced in United States

Cy5-conjugated donkey anti-mouse IgG is a secondary antibody used for the detection of mouse immunoglobulin G (IgG) in various immunoassays. The antibody is conjugated with the fluorescent dye Cyanine 5 (Cy5), allowing for visualization and quantification of target proteins.

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Anti-mouse Cy5 (30 citations)

5 protocols using cy5 conjugated donkey anti mouse igg

Radioligand Binding Assay Protocol

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5-HT (5-HT hydrochloride), clozapine (8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine), mesulergine (N′-[(8a)-1,6-dimethylergolin-8-yl]-N,N-dimethylsulfamide hydrochloride), metergoline ([[(8β)-1,6-dimethylergolin-8-yl]-methyl]carbamic acid phenylmethyl ester), isoprenaline hydrochloride, chloroquine (N⁴-(7-chloro-4-quinolinyl)-N¹,N¹-dimethyl-1,4-pentanediamine diphosphate) and mouse anti-FLAG M2 were from Sigma-Aldrich (St. Louis, MO, USA). SB269970 ((R)-3-(2-(2-(4-methyl-piperidin-1-yl)ethyl)-pyrrolidine-1-sulphonyl)-phenol) was from Tocris (Bristol, UK). Olanzapine (ad injectabila) was from Eli Lilly & Co. (Indianapolis, IN, USA). G418, penicillin-streptomycin, L-glutamine and LipofectAMINE2000™ were from Life Technologies). Cy5-conjugated donkey anti-mouse IgG was from Jackson ImmunoResearch (West Grove, PA, USA). [³H]-5-CT (5-carboxamidotryptamine) (60–102 Ci·mmol⁻¹), [N⁶-methyl-³H]-mesulergine (87 Ci·mmol⁻¹), [³H]-SB269970 (36 Ci·mmol⁻¹) and [³H]-CGP12177 (37 Ci·mmol⁻¹) were from GE Healthcare (Buckinghamshire, UK).

Andressen K.W., Manfra O., Brevik C.H., Ulsund A.H., Vanhoenacker P., Levy F.O, & Krobert K.A. (2015). The atypical antipsychotics clozapine and olanzapine promote down-regulation and display functional selectivity at human 5-HT7 receptors. British Journal of Pharmacology, 172(15), 3846-3860.

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Mapping Refeeding-Activated Neurons in the Brain

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To identify the brain regions where refeeding-activated neurons are contacted by PB neurons, triple-labeling immunofluorescence for PHA-L, c-Fos and the neuronal marker, HuC/HuD, was performed on sections of all animals (n=5) with a PHA-L injection site confined within the area of the PB. Following pre-treatment as described above, the sections were incubated in anti-PHA-L serum at 1:5,000 for 2 days at room temperature, followed by biotinylated donkey anti-rabbit IgG (Jackson Immunoresearch) diluted to 1:500 and then ABC (1:1,000) for 1 h after rinses in PBS. The immunoreaction was amplified with biotinylated tyramide as above, and after further washes, the sections were incubated in streptavidin-conjugated Alexa 555 (Vector) at a 1:500 dilution. Then, the sections were incubated in a mixture of rabbit antiserum against c-Fos at 1:2,000 dilution and mouse antiserum against HuC/HuD (Molecular Probes) at 1:500 for 2 days at 4°C. After washes in PBS, the sections were immersed in a cocktail of fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (Jackson) at 1:250 dilution and Cy5-conjugated donkey anti-mouse IgG (Jackson) at 1:100 dilution and incubated for 2 h at room temperature. The sections were then mounted onto glass slides and coverslipped with Vectashield Mounting Medium.

Zséli G., Vida B., Martinez A., Lechan R.M., Khan A.M, & Fekete C. (2016). Elucidation of the Anatomy of a Satiety Network: Focus on Connectivity of the Parabrachial Nucleus in the Adult Rat. The Journal of comparative neurology, 524(14), 2803-2827.

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Immunofluorescence Labeling of Paraffin Tissue

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For immunofluorescence labeling using paraffin embedded tissue sections the treatment procedure was performed as previously described^{8 (link),22 (link),88 (link)}. In sum, after deparaffinizing and buffering, the slides were incubated with a mixture of the rabbit IL-6 antibody (see above) and mouse anti-MAP2 (microtubule-associated protein 2, 1:200; Millipore, Temecula, USA) as well as mouse anti-GFAP (1:1000; Sigma-Aldrich, St. Louis, USA) in 5% fetal calf serum in buffer over night at 4 °C. The primary antibodies were visualized with Cy2-conjugated donkey anti-rabbit (1:400) and Cy5-conjugated donkey anti-mouse IgG (1:200; both Jackson ImmunoResearch, West Grove, USA) after 2 h incubation. Further, slides were stained with Hoechst 33342 (1:1000, Molecular Probes, Leiden, Netherlands) to identify the cell nuclei by auto-fluorescence utilizing an ultraviolet laser (362 nm). Strict internal control runs were done without primary antibodies.
The immunofluorescence was investigated by a confocal laser scanning microscope (Leica SP8 confocal microscope; Leica, Wetzlar, Germany) using excitation wavelengths of 488 nm (argon laser, yellow-green Cy2-immunofluorescence labelling), 543 nm (helium/neon1, red Cy3-immunofluorescence) and 633 nm (helium/neon2, blue Cy5-labelling).

Trautz F., Franke H., Bohnert S., Hammer N., Müller W., Stassart R., Tse R., Zwirner J., Dreßler J, & Ondruschka B. (2019). Survival-time dependent increase in neuronal IL-6 and astroglial GFAP expression in fatally injured human brain tissue. Scientific Reports, 9, 11771.

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Confocal Microscopy for Immunofluorescence Analysis

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For confocal microscopy, detection of immunofluorescence-labeled antigens utilized two combinations of secondary antibodies. The first combination included secondary antibodies that were fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (1:200; Jackson Immunoresearch) for MGL or FAAH detection; tetramethylrhodamine-5-isothiocyanate (TRITC)-conjugated donkey anti-guinea pig IgG (1:200; Jackson Immunoresearch) for CB1 detection and Cy5-conjugated donkey anti-mouse IgG (1:200; Jackson Immunoresearch) for DβH or NET detection. Tissue sections underwent serial dehydration, were mounted on slides, and coverslipped using DPX (Aldrich). Slides were then viewed using an Olympus IX81 inverted confocal microscope (Hatagaya, Shibuya-Ku, Tokyo, Japan), with helium and argon laser excitation wavelengths of 488, 543 and 635. The microscope is also equipped with filters (DM 405–44, BA505-605, and BA 560–660) and the Olympus Fluoview ASW FV1000 program. Fluorescence confocal images were assembled and adjusted for brightness and contrast in Adobe Photoshop.

Urquhart M.A., Ross J.A., Reyes B.A., Nitikman M., Thomas S.A., Mackie K, & Van Bockstaele E.J. (2019). Noradrenergic depletion causes sex specific alterations in the endocannabinoid system in the Murine prefrontal cortex. Neurobiology of Stress, 10, 100164.

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Quantifying Serotonergic Neuron Loss in RVM

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Previous studies using transient markers such as TUNEL or caspase-3 have not successfully demonstrated apoptosis in the RVM after peripheral nerve injury [32 (link), 44 (link)], possibly due to the very short duration of apoptosis [34 (link)]. For that reason we used neuronal counting, a cumulative method for determining cell loss that is not sensitive to the speed at which neurons die. The RVM was sectioned using a freezing microtome (Leica, SM2400) at a nominal thickness of 50 μm. The free-floating sections were washed in phosphate-buffered saline (PBS) for three 5-minute intervals. To identify serotonergic neurons, we stained for tryptophan hydroxylase (TPH), the enzyme catalyzing the rate-limiting step in serotonin synthesis. RVM sections were incubated overnight at 4°C in a solution containing mouse anti-TPH; (catalog #T0678, Sigma, Saint Louis, MO, 1:1000). Sections were then washed in PBS and incubated for 4 h with Cy5-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, 1:500). All sections were counterstained with the fluorescent Nissl stain ethidium bromide (30 nM, Sigma; [39 (link)]) [39 (link)].

Leong M.L., Speltz R, & Wessendorf M. (2016). Effects of chronic constriction injury and spared nerve injury, two models of neuropathic pain, on the numbers of neurons and glia in the rostral ventromedial medulla. Neuroscience letters, 617, 82-87.

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