Donkey anti rat igg cy3

Manufactured by Jackson ImmunoResearch

Donkey anti-rat IgG-Cy3 is a secondary antibody produced in donkeys and conjugated to the Cyanine 3 (Cy3) fluorescent dye. It is designed to detect and visualize rat immunoglobulin G (IgG) in various immunoassays and imaging applications.

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Anti-rat Cy3 (13 citations) Anti-rat IgG Cy3 (6 citations)

11 protocols using donkey anti rat igg cy3

Comprehensive Antibody Immunoassay Protocol

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The following antibodies were used for immunofluorescence or flow cytometry assays: mouse anti‐human CD11c monoclonal antibody (cat# 14‐0116‐82, Thermo Fisher Scientific); rat anti‐mouse Lamp‐1 antibody (clone 1D4B, Developmental Studies Hybridoma Bank); mouse anti‐human Lamp‐1 antibody (clone H4A3, Developmental Studies Hybridoma Bank); mouse anti‐human GM130 antibody (cat# 610822, BD Biosciences); rabbit anti‐human calnexin antibody (cat# ab112995, abcam); rabbit anti‐bordetella pertussis toxin antibody (cat# ab188414, abcam); rabbit anti‐human TGN46 antibody (cat# ab50595, abcam); rabbit anti‐cathepsin D antibody (cat# 219361, Millipore Sigma); Cy3‐donkey anti‐rat IgG (cat# 712–165‐153, Jackson Immunoresearch); donkey anti‐rabbit Alexa Fluor 647 (cat# A31573, Invitrogen); donkey anti‐rabbit IgG Alexa Fluor 555 (cat# A31572, Life Technologies); goat anti‐mouse Alexa Fluor 488 (cat# 11029, Thermo Fisher Scientific); goat anti‐rabbit Alexa Fluor 647 (cat# A21245, Thermo Fisher Scientific); goat anti‐mouse Alexa Fluor 647 (cat# A21235, Thermo Fisher Scientific); donkey anti‐mouse Alexa Fluor 555 (cat# A31570, Thermo Fisher Scientific). The following antibodies were used during blocking and opsonization assays: rat anti‐mouse CD11b (blocking antibody, clone: M1/70, cat# AB‐467108, Thermo Fisher Scientific) and human IgG (opsonization, I8640 or I4506, Millipore Sigma).

Jaldin‐Fincati J.R., Moussaoui S., Gimenez M.C., Ho C.Y., Lancaster C.E., Botelho R.J., Ausar S.F., Brookes R.H, & Terebiznik M.R. (2022). Aluminum hydroxide adjuvant diverts the uptake and trafficking of genetically detoxified pertussis toxin to lysosomes in macrophages. Molecular Microbiology, 117(5), 1173-1195.

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Corneal Angiogenesis Assay in Mice

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The corneal micropocket assay was performed as previously described²³. In summary, a lamellar micropocket was dissected (towards the temporal limbus) in the eyes of 8 week-old female C57BL6 mice. In this pocket on the corneal surface, a pellet was inserted containing 80 ng recombinant human basic fibroblast growth factor (bFGF, PeproTech) and 100 ng of a negative control siRNA NC5, of Pck2-targeting siRNA mm.Ri.Pck2.13.3 (siPck2 #1) or mm.Ri.Pck2.13.4 (siPck2 #2) (IDT Integrated DNA Technologies). Seven days later, mice were euthanized, eyes were enucleated and corneas were isolated and fixed with ethanol (70%) for whole-mount CD31 antibody staining (primary antibody: rat anti-mouse CD31 MEC 13.3, BD Biosciences 557355, dilution 1:200; secondary antibody: Cy3-donkey anti-rat IgG, Jackson ImmunoResearch 712-165-153, dilution 1:200). After imaging the flat-mounted corneas, the CD31⁺ area was quantified (ImageJ) as percentage of total cornea area. Animal housing and all experimental procedures were approved by the Institutional Animal Ethics Committee of the KU Leuven (Belgium) under protocols P042/2020.

de Zeeuw P., Treps L., García-Caballero M., Harjes U., Kalucka J., De Legher C., Brepoels K., Peeters K., Vinckier S., Souffreau J., Bouché A., Taverna F., Dehairs J., Talebi A., Ghesquière B., Swinnen J., Schoonjans L., Eelen G., Dewerchin M, & Carmeliet P. (2024). The gluconeogenesis enzyme PCK2 has a non-enzymatic role in proteostasis in endothelial cells. Communications Biology, 7, 618.

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Lysosome Localization Analysis in Cells

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Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized by adding 0.1% of Triton buffer. Then, 5% of normal goat serum (NGS) was used to block the unspecific unions. Primary antibodies (mouse monoclonal anti-ApoE (sc53570, Santa Cruz Biotechnology, dilution 1:400) and rat monoclonal anti-Lamp1 (1D4B, Hybridoma Bank, dilution 1:200) were incubated overnight, followed by one-hour incubation with the secondary antibody Cy3-donkey anti-rat IgG (712–165-150, Jackson Immunoresearch, dilution 1:200) and Alexa fluor 488 goat anti-mouse IgG (A11029, Thermo Fisher Scientific, dilution 1:1000). Coverslips were mounted on a slide with Fluoromount G. Images were acquired with confocal laser scanning microscopy ZEISS LSM 700. Analysis of lysosome localization was carried out using the ImarisCell tool of IMARIS software (Bitplane), which permits manual selection of the nucleus and the membrane of each cell and computes the distance (μm) from each Lamp1-positive vesicle to the nucleus center. Lamp1-positive vesicles larger than 0.5 μm were considered. The frequency distribution of vesicles from the nucleus center to the cellular membrane was represented.

Larramona-Arcas R., González-Arias C., Perea G., Gutiérrez A., Vitorica J., García-Barrera T., Gómez-Ariza J.L., Pascua-Maestro R., Ganfornina M.D., Kara E., Hudry E., Martinez-Vicente M., Vila M., Galea E, & Masgrau R. (2020). Sex-dependent calcium hyperactivity due to lysosomal-related dysfunction in astrocytes from APOE4 versus APOE3 gene targeted replacement mice. Molecular Neurodegeneration, 15, 35.

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Immunocytochemistry of Bone Marrow-Derived Macrophages

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Bone marrow was plated on chamber slides (Lab-Tek) and differentiated into BMDMs as described above. On day 7, slides were fixed with 4% PFA for 10 min, permeabilized with 0.5% saponin for 10 min, blocked [1% BSA, 22.52 mg/ml glycine in PBS with 0.1% Tween 20 (PBST)] for 1 h and incubated in primary antibody diluted in 1% BSA in PBST overnight at 4°C. After washing with PBS, slides were incubated in secondary antibody with Hoechst (20 µg/ml) in 1% BSA in PBST for 1 h. After mounting with DABCO, images were obtained with a Nikon Eclipse Ti-E microscope. Primary antibodies used for immunocytochemistry were: rabbit anti-CD11b (1:250; #EPR1344; Abcam) and rat anti-CD68 (1:250; #MCA1957; BioRad). Secondary antibodies used were acquired from Jackson ImmunoResearch and used at a 1:500 dilution: Cy3 donkey anti-rat IgG (#712-165-153) and Cy5 donkey anti-rabbit IgG (#711-175-152).

Colijn S., Muthukumar V., Xie J., Gao S, & Griffin C.T. (2020). Cell-specific and athero-protective roles for RIPK3 in a murine model of atherosclerosis. Disease Models & Mechanisms, 13(1), dmm041962.

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Aortic Root Macrophage and Smooth Muscle Quantification

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Macrophages and smooth muscle cells of the aortic root were visualized using rat anti-CD68 (1:250; #MCA1957; Bio-Rad) and Cy3-conjugated anti-aSMA (1:250; #C6198; Sigma-Aldrich) antibodies, respectively. Sections were blocked with 5% bovine serum albumin (BSA) for 2 h, incubated with primary antibody overnight, and incubated with secondary antibody Cy3 donkey anti-rat IgG (1:500; Jackson ImmunoResearch) for CD68 and Hoechst for nuclei (20 µg/ml) for 1 h. For macrophages, three aortic root sections per mouse were analyzed at 120 μm apart. For smooth muscle cells, one aortic root section per mouse was analyzed from the middle of the root. The average CD68⁺ area or aSMA⁺ area was reported as the percent area of the total lesion area.

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Immunofluorescence Staining of NF-κB Pathway

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Anti-mouse NF-κB p65 antibody (Ab7970; Abcam, Cambridge, UK) and anti-mouse B220 (14-0452-81; eBioscience, San Diego, CA) were used as primary antibodies, and donkey anti-rat IgG-Alexa Fluor 488 [712-545-153; Jackson ImmunoResearch Laboratories Inc. (JIR), West Grove, PA], donkey anti-rabbit IgG-Alexa Fluor 488 (711-545-152; JIR), donkey anti-rat IgG-Cy3 (712-165-153; JIR) and donkey anti-rabbit IgG-Cy3 (711-165-152; JIR) were used as secondary antibodies. Staining was conducted as described previously [47] (link). Briefly, antigen retrieval was performed in a steam pressure cooker with prewarmed antigen retrieval buffer, citrate pH 6 (S203130; Dako, Glostrup, Denmark) at 95°C for 15 min. After blocking with 3% bovine serum albumin in phosphate-buffered saline, sections (4 μm) were incubated with anti-NF-κB, -Iκ-B, -B200 or -A20 antibodies at a 1∶200 dilution each at 4°C overnight. Sections were incubated with secondary antibodies and anti-rat Alexa Fluor 488, -rabbit Alexa Fluor 488, -rat Alexa Fluor 546, and -rabbit Alexa Fluor 546 at room temperature for 2 hours. Nuclei were stained with Hoechst 333421 (H3570; Life Technologies).

Kasama Y., Mizukami T., Kusunoki H., Peveling-Oberhag J., Nishito Y., Ozawa M., Kohara M., Mizuochi T, & Tsukiyama-Kohara K. (2014). B-Cell-Intrinsic Hepatitis C Virus Expression Leads to B-Cell-Lymphomagenesis and Induction of NF-κB Signalling. PLoS ONE, 9(3), e91373.

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Immunohistochemical Characterization of Neural Markers

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Mice were sacrificed and brain sections were prepared as previously reported (Deng, 2013 (link)). Primary antibodies used were: rat anti-BrdU (1:250, AbD Serotec CAT# OBT0030CX), mouse anti-NeuN (1:100, Millipore (CHEMICON/Upstate/Linco) CAT# MAB377), and goat anti-DCX (1:250, Santa Cruz Biotechnology CAT# sc-8066). All secondary antibodies were from Jackson ImmunoResearch (Donkey anti-rat IgG Cy3 CAT# 712-165-153, donkey anti-mouse IgG Alexa Fluor 647 CAT# 715-605-151, donkey anti-goat IgG Alexa Fluor 488 CAT# 705-545-147) at 1:250 dilutions. DAPI (Sigma–Aldrich CAT# D9542) was used to stain individual cells.

Clemenson G.D., Lee S.W., Deng W., Barrera V.R., Iwamoto K.S., Fanselow M.S, & Gage F.H. (2014). Enrichment Rescues Contextual Discrimination Deficit Associated With Immediate Shock. Hippocampus, 25(3), 385-392.

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Staining and Imaging Notch Signaling

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Wing discs from wandering larvae or ovaries and brain from adult flies were dissected in 1x PBS and fixed for 30 minutes in 4% paraformaldehyde in PBS. Tissues were then washed with 0.2% PBST for total Notch staining or with cell-culture grade PBS (Thermo Fisher Scientific, #10010023) for extracellular Notch staining. All antibodies used in this study are listed in S1 Table. Primary antibodies [mouse anti-Notch intracellular domain (NICD) (1:50; DSHB, C17.9C6), mouse anti-Cut (1:100; DSHB, 2B10), rat anti-HA (1:100, Sigma-Aldrich, 11867423001), or mouse anti-NECD (1:100; DSHB, C458.2H)] were applied in 0.2% PBST or PBS with 5% NDS/0.1% NaN₃ overnight at 4°C. Tissue was then washed with 0.2% PBST or PBS (3 times, 15 minutes each) and secondary antibodies/stains [donkey anti-rat IgG-Cy3 (1:500; Jackson ImmunoResearch #712-165-153), donkey anti-mouse IgG-Alexa-647 (1:500; Jackson ImmunoResearch #715-605-151), donkey anti-mouse Alexa-488 (1:500; Jackson ImmunoResearch #715-545-151), and/or Alexa-488 Phalloidin (1:1000; ThermoFisher A12379)] were applied in 0.2% PBST+NDS or PBS+NDS for 2 hours at room temperature. Tissues were further washed with 0.2% PBST or PBS and mounted in Vectashield with DAPI (Vector labs). Images were taken with LSM 710 Confocal Microscope (Zeiss).

Salazar J.L., Yang S.A., Lin Y.Q., Li-Kroeger D., Marcogliese P.C., Deal S.L., Neely G.G, & Yamamoto S. (2021). TM2D genes regulate Notch signaling and neuronal function in Drosophila. PLoS Genetics, 17(12), e1009962.

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Fluorescence Immunostaining of Retinal Cells

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Retinas were prepared as described for IB₄-Alexa 594 staining, and incubated with primary antibodies in RSB at 4°C overnight. Primary antibodies included rabbit anti-GFAP (1∶200, Life Technologies), rat anti-GFAP (2 µg/ml, Life Technologies), rabbit anti-Pax2 (1 µg/ml, Life Technologies), and goat anti- PDGFRα (1 µg/ml, R&D Systems). For Pax2 and GFAP double IF staining, rabbit anti-Pax2 and rat anti-GFAP were used. Following incubation with primary antibodies, retinas were washed, and incubated overnight with appropriate secondary antibodies including goat anti-rabbit IgG-Alexa fluor®-488 (1∶200, Life Technologies), donkey anti-rat IgG-Cy3 (2 µg/ml, Jackson ImmunoResearch, , West Grove, PA), and donkey anti-goat IgG-Alexa fluor®-488. Stained retinas were washed thoroughly, and mounted in 50% glycerol in PBS. Images were taken with a Zeiss LSM 510 confocal microscope.

Duan L.J., Takeda K, & Fong G.H. (2014). Hypoxia Inducible Factor-2α Regulates the Development of Retinal Astrocytic Network by Maintaining Adequate Supply of Astrocyte Progenitors. PLoS ONE, 9(1), e84736.

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Immunofluorescence Staining of Cilia and Stem Cell Markers

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Cells were fixed in 4% paraformaldehyde for 6 min at room temperature. The cells were then processed for immunofluorescence using mouse antibody against β-Tubulin IV (1:200, Sigma-Aldrich, T7941) to stain cilia. Rabbit anti-FoxJ1 polyclonal antibody (1:200, Sigma, HPA005714), mouse anti-nestin monoclonal antibody (1:400, BD, BD611658), and rat anti-BrdU monoclonal antibody (1:200, AbD serotec, OBT0030) were also used as primary antibodies. Donkey anti-mouse IgG-Alexa488 (1:500, Jackson ImmunoResearch, 715-545-150), donkey anti-rabbit IgG-Cy3 (1:500, Jackson ImmunoResearch, 712-165-153), and donkey anti-rat IgG-Cy3 (1:500, Jackson ImmunoResearch, 711-165-152) were used as secondary antibodies. Nuclei were counterstained with DAPI. Fluorescent images of cells were obtained using a fluorescent microscope (BZ-X710, Keyence). The total number of cells was determined by counting DAPI stained nuclear signals using Fiji (ImageJ) (Schindelin et al. 2012) . The number of FoxJ1 positive cells was also determined using Fiji. Numbers of BrdU or nestin-positive cells or multi-ciliated cells were counted manually.

Hiraoka K., Inada H., Yanai K, & Osumi N. (2020). Bone Morphogenetic Proteins Inhibit Ciliogenesis of Ependymal Cells in Vitro. The Tohoku journal of experimental medicine, 252(3).

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