Cy3 conjugated anti rabbit secondary antibody

Manufactured by Jackson ImmunoResearch

Sourced in United States, Panama

Cy3-conjugated anti-rabbit secondary antibody is a laboratory reagent used in immunoassays and other biological applications. It is designed to bind to and detect primary antibodies raised in rabbit, enabling visualization and quantification of target analytes.

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Lab products found in correlation

Cy3 conjugated anti mouse secondary antibody, by Jackson ImmunoResearch (3 mentions) Hoechst 33342, by Merck Group (3 mentions) 4 6 diamidine 2 phenylindole dapi, by Merck Group (2 mentions) Fitc conjugated anti mouse secondary antibody, by Jackson ImmunoResearch (2 mentions) Rabbit polyclonal anti aif 1 antibody, by Thermo Fisher Scientific (2 mentions) Lsm 510, by Zeiss (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Anti nrf2, by Santa Cruz Biotechnology (1 mentions) P21cip1, by Santa Cruz Biotechnology (1 mentions) Anti rfp, by Thermo Fisher Scientific (1 mentions) Fw3000, by Olympus (1 mentions) Anti α tubulin, by Merck Group (1 mentions) Rat anti cd31 antibody, by BD (1 mentions) Rabbit anti cd68 antibody, by Abcam (1 mentions) Rabbit anti pbr antibody, by Abcam (1 mentions) Bx ucb, by Olympus (1 mentions) Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions) Dapagliflozin, by Cayman Chemical (1 mentions) Penicillin streptomycin, by Sartorius (1 mentions) Rapamycin, by Cell Signaling Technology (1 mentions)

26 protocols using cy3 conjugated anti rabbit secondary antibody

Immunofluorescence Analysis of Mitotic Index in PnM Hybrid Cells

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PnM cells were fixed with 4% formaldehyde for 20 min, following previously published protocols^{67 (link)}. In order to determine the mitotic index for the PnM hybrid clone, a primary antibody against phosphohistone H3 (P-H3; rabbit used at 1:100; Epitomics) was used for immunofluorescence in a 1× PBS buffer. A Cy3-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories) was used at 1:100. Mitotic index for PnM cells was determined to be 2.48% ± 0.78, N = 1300.
In order to determine cell type as described previously^{71 (link)}, PnM were immunostained using rabbit anti-dMef2 antibody, which was a gift from Bruce Paterson^{72 (link)}. The antibody was used at 1:1000, with a Cy3-conjugated anti-rabbit secondary antibody at 1:100 (Jackson immunoresearch). The cells expressed dMef2 exclusively, and are most likely of mesodermal origin, as they tested negative for other cell type markers including an epithelial cell marker; D-E-Cadherin, ((Rat)-anti E-Cadherin 1:5 (Hybridoma Bank, Iowa), a fat cell marker; Nile red solution, (Sigma; 1% stock in DMSO diluted to 1:5000), and a nerve cell marker; horse Radish peroxidase (HRP) (Jackson immunoresearch (Rhodamine conjugated) 1:200).

AlHaj Abed J., Erceg J., Goloborodko A., Nguyen S.C., McCole R.B., Saylor W., Fudenberg G., Lajoie B.R., Dekker J., Mirny L.A, & Wu C.T. (2019). Highly structured homolog pairing reflects functional organization of the Drosophila genome. Nature Communications, 10, 4485.

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Nrf2, Plk2, and p21 Protein Localization

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NRK52E cells grown on coverslips were fixed with 4% paraformaldehyde. Methanol was used for the permeabilization. Cells were washed three times with 0.1% Triton X-100 in phosphate-buffered saline (PBS), incubated overnight at 4 °C in PBS containing 0.1% Triton X-100 and 3% bovine serum albumin to block nonspecific interactions, and then incubated with anti-Nrf2 (Santa Cruz Biotechnology, sc-13032), Plk2 (Santa Cruz Biotechnology, sc-374643), p21^cip1 (Santa Cruz Biotechnology, sc-6246), and p21^cip1 (MyBioSource, San Diego, CA, USA; MBS440016) antibodies. The cells were washed three times with PBST (0.1% Triton X-100) and then incubated with fluorescein isothiocyanate (FITC)–conjugated anti-rabbit secondary antibodies (Invitrogen), fluorescein isothiocyanate (FITC)–conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories; West Grove, PA, USA), Cy3-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories), or Cy3-conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories) and 4′, 6-diamidine-2-phenylindole (DAPI) (Sigma-Aldrich) for staining nuclear DNA. Images of cells were collected and evaluated with a confocal microscope FW3000 (Olympus; Tokyo, Japan).

Kim D.E., Byeon H.E., Kim D.H., Kim S.G, & Yim H. (2022). Plk2-mediated phosphorylation and translocalization of Nrf2 activates anti-inflammation through p53/Plk2/p21cip1 signaling in acute kidney injury. Cell Biology and Toxicology, 39(4), 1509-1529.

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Immunofluorescence Staining of A549 Cells

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A549 cells grown on coverslips were fixed with 4% paraformaldehyde. Methanol was used for permeabilization. Cells were washed three times with 0.1% Triton X-100 in PBS (PBST), incubated overnight at 4°C in PBST and 3% bovine serum albumin (BSA), and then incubated with anti-RFP (Life Technologies, R10367) and anti-α-tubulin (Sigma-Aldrich, MABT205) antibodies. The cells were washed three times with PBST and then incubated with Cy3-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), FITC-conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch Laboratories), and 4′, 6-diamidine-2-phenylindole (DAPI) (Sigma-Aldrich) for DNA staining. Images of cells were collected and evaluated with a confocal microscope FW3000 (Olympus; Tokyo, Japan).

Kim D.E., Shin S.B., Kim C.H., Kim Y.B., Oh H.J, & Yim H. (2023). PLK1-mediated phosphorylation of β-catenin enhances its stability and transcriptional activity for extracellular matrix remodeling in metastatic NSCLC. Theranostics, 13(3), 1198-1216.

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Inflammation and Tumor Tissue Immunohistochemistry

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The inflammatory muscles of mice on day 1, 6 and 12 after turpentine injection and A549, HT29, U87MG, INS-1, 4T1 tumors was collected and embedded and frozen in CRYO-OCT compound. Cryosecitons with thickness of 10 μm were fixed in Z-Fix for 10 minutes, then rinsed with PBS and blocked with 2% bovine serum albumin (BSA) for 30 minutes at room temperature. Inflammation muscle slices were incubated with rabbit anti-CD68 antibody (1:100; Abcam, USA) or rat anti-CD31 antibody (1:100, BD Biosciences) or rabbit anti-PBR antibody (1:100; Abcam, USA) over night at 4 ºC. After 10 min PBS wash for 3 times, slices were incubated with Cy3-conjugated anti-rabbit or FITC-conjugated anti-rat secondary antibodies (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at dark room in room temperature followed by 10 min PBS wash for 3 times. Tumor tissue sections were incubated with rabbit anti-PBR antibody (1:100; Abcam, USA) for 1 h and then with Cy3-conjugated anti-rabbit secondary antibodies (1:200; Jackson ImmunoResearch Laboratories) for 30 min at dark room in room temperature followed by 10 min PBS wash for 3 times. All slices were mounted with VECTASHIELD^® mounting medium containing DAPI and covered with cover slides before visualization under an epifluorescence microscopy (IX-81, Olympus).

Wu C., Yue X., Lang L., Kiesewetter D.O., Li F., Zhu Z., Niu G, & Chen X. (2014). Longitudinal PET Imaging of Muscular Inflammation Using 18F-DPA-714 and 18F-Alfatide II and Differentiation with Tumors. Theranostics, 4(5), 546-555.

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Mitochondria and Autophagy Visualization

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Fibroblasts grown on glass coverslips were incubated with 2 μM MitoTracker Red CMXRos (Invitrogen) for 45 min at 37°C, 5% CO2. Cells were washed in PBS and fixed with 4% paraformaldehyde for 15 min and then permeabilized for 10 min with 0.3% Triton X-100. Following, blocking was performed with 2.5% bovine serum albumin in PBS for 30 min. Subsequently, cells were incubated with primary antibodies directed against LC3B (microtubule-associated protein 1 light chain-3B, Cell signaling) for 2 h at room temperature. After washing, they were incubated for 1 h at room temperature with Cy3-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA), washed, mounted on slides and examined with a confocal microscope (Olympus BX-UCB).

Kogot-Levin A., Saada A., Leibowitz G., Soiferman D., Douiev L., Raz I, & Weksler-Zangen S. (2016). Upregulation of Mitochondrial Content in Cytochrome c Oxidase Deficient Fibroblasts. PLoS ONE, 11(10), e0165417.

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Glucose and SGLT2i Regulate S6 Phosphorylation

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LLC-PK1 and HK-2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 IU/mL penicillin/streptomycin (Biological Industries) at 37 °C in a humid atmosphere with 5% CO₂. For studying S6 phosphorylation, cells were starved in serum-free DMEM for 1 h and then exposed to either 5 mM or 30 mM D-glucose (low and high glucose, respectively) in the presence or absence of SGLT2i (dapagliflozin; 5 μM, Cayman Chemical) for 0.5 h. For immunofluorescence, cells were exposed to either 5 mM or 30 mM D-glucose in the presence or absence of SGLT2i (5 μM) for 48 h. Rapamycin and Torin-1 (Cell Signaling) were added to the medium at a concentration of 100 and 250 nM, respectively. For inhibiting system L amino acid transporters, cells were treated with 20 mM BCH (Sigma-Aldrich) for 24 h.
For immunofluorescence, cells were grown on glass coverslips and fixed with 4% paraformaldehyde. After permeabilization and blocking, cells were incubated with anti-pS6 antibodies (5364S, Cell Signaling) followed by incubation with Cy3-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories). Cells were mounted on slides and examined with a Nikon A1R confocal microscope.

Kogot-Levin A., Hinden L., Riahi Y., Israeli T., Tirosh B., Cerasi E., Mizrachi E.B., Tam J., Mosenzon O, & Leibowitz G. (2020). Proximal Tubule mTORC1 Is a Central Player in the Pathophysiology of Diabetic Nephropathy and Its Correction by SGLT2 Inhibitors. Cell Reports, 32(4), 107954.

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Immunofluorescence Staining of Cx26 in Cryosections

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Skin was fixed in a 1% formaldehyde in PBS for 1 h at room temperature. Tissues were rinsed with PBS, immersed in Optimal Cutting Temperature compound (Ted Pella, Redding, CA), and frozen. 8–10 μm sections were cut on a cryotome, dried onto glass slides, and stained with polyclonal rabbit antibodies against Cx26 (Zymed, San Francisco, CA), washed with 0.1% Triton X-100/PBS, incubated with Cy3-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch, West Grove, PA), washed with 0.1% Triton X-100/PBS, and mounted using Vectashield with 4′,6-diamidino-2-phenylindole (Vector, Burlingame, CA).

Sellitto C., Li L, & White T.W. (2021). Connexin hemichannel inhibition ameliorates epidermal pathology in a mouse model of keratitis ichthyosis deafness syndrome. Scientific Reports, 11, 24118.

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Immunofluorescence Staining of Nucleolin

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U2-OS cells were seeded on glass slides in a 24-well plate (5 × 103 cells/well) and transfected with GFP-fused Cas9 plasmid using FuGENE (Promega, Wisconsin, USA) after cell adherence. At 72 h after transfection, the slides were gently washed with PBS and fixed in 4% paraformaldehyde, then permeabilized in 0.5% Triton X-100 to perforate nuclear membranes. After blocking with 4% BSA, the samples were incubated with anti-NCL antibody (1:1000) (Proteintech, Illinois, USA) in PBS that contained 4% BSA at 4 °C overnight or at 37 °C for 1 h. They were then stained with Cy3-conjugated anti-rabbit secondary antibody (Jackson Immunoresearch, Pennsylvania, USA) at 37 °C for 30 min. Following extensive washing, the slides were coated with mounting medium (ThermoFisher). Cell fluorescence was visualized under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Tan R., Du W., Liu Y., Cong X., Bai M., Jiang C., Li Z., Tan M., Ma D.K., Huang Q., Jiang W, & Dang Y. (2020). Nucleolus localization of SpyCas9 affects its stability and interferes with host protein translation in mammalian cells. Genes & Diseases, 9(3), 731-740.

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Quantifying Synaptic Protein Expression in the PRC

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One out of four sections (3–6 per mouse) of the PRC was used for immunohistochemistry. Free-floating sections were stained overnight at 4°C with the primary antibodies post-synaptic density protein 95 (1:1000, anti-PSD-95 rabbit polyclonal Ab, Abcam, Cambridge, United Kingdom) or glutamate vesicular transporter 1 (1:500, anti-VGlut1 rabbit polyclonal Ab, Thermo Scientific), and then stained in fluorescent secondary antibody (Cy3-conjugated anti-rabbit secondary antibody 1:200; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, United States) for 2 h at room temperature. For quantification of synaptic puncta, images immunoprocessed for PSD-95 and VGlut1 were acquired with a Leica TCS SL confocal microscope. In each section four images from the PRC were captured and the density of individual puncta exhibiting VGlut1 or PSD-95 immunoreactivity was evaluated as previously described (Guidi et al., 2013 (link)).

Ren E., Roncacé V., Trazzi S., Fuchs C., Medici G., Gennaccaro L., Loi M., Galvani G., Ye K., Rimondini R., Aicardi G, & Ciani E. (2019). Functional and Structural Impairments in the Perirhinal Cortex of a Mouse Model of CDKL5 Deficiency Disorder Are Rescued by a TrkB Agonist. Frontiers in Cellular Neuroscience, 13, 169.

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Dacomitinib Modulates EGFR Phosphorylation

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U87vIII.Luc2 cells were seeded onto glass slides and cultured overnight in medium containing 10% FBS. Cells were treated with DMSO (0.01%) or 1 μM dacomitinib for 16 hours prior to fixation in 4% paraformaldehyde in PBS. Cells were blocked and permeabilized with PBS containing 0.1% Triton X-100 and 10% normal goat serum (Vector Laboratories) at room temperature for 1 hour. Cells were further incubated with an anti-phosphorylated EGFR Y1173 primary antibody (Cell Signaling #4407, 1:200) followed by a Cy3-conjugated anti-rabbit secondary antibody (Jackson Immunoresearch, 1:400). Nuclei counterstained with DAPI and slides coverslipped using VectorShield HardSet mounting medium (Vector Laboratories). Wide-field epifluorescence images were taken using a Nikon Ti Eclipse and NIS Elements software (Nikon).

Endersby R., Whitehouse J., Hii H., Greenall S.A., Johns T.G, & Gottardo N.G. (2018). A Pre-Clinical Assessment of the Pan-ERBB Inhibitor Dacomitinib in Pediatric and Adult Brain Tumors. Neoplasia (New York, N.Y.), 20(5), 432-442.

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