Cy3 conjugated secondary antibody

Manufactured by Proteintech

Sourced in United States, China

Cy3-conjugated secondary antibody is a fluorescent-labeled antibody that binds to the primary antibody. It can be used to detect and visualize target proteins in various applications such as Western blotting, immunohistochemistry, and flow cytometry.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Anti troponin 1, by Santa Cruz Biotechnology (2 mentions) Zoetm fluorescent cell imager, by Bio-Rad (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions) Fluorescence conjugated secondary antibody, by Proteintech (1 mentions) Anti cd133, by Proteintech (1 mentions) Anti gfap, by Proteintech (1 mentions) Poly l ornithine, by BD (1 mentions) Lsm 710, by Zeiss (1 mentions) Fitc conjugated secondary antibody, by Proteintech (1 mentions) Ckx41, by Olympus (1 mentions) Anti ampk, by Cell Signaling Technology (1 mentions) Hoechst, by Merck Group (1 mentions) A1 confocal microscope, by Nikon (1 mentions) 4 6 diamidino 2 phenylindole dapi, by Bioss Antibodies (1 mentions) Anti ki67, by Proteintech (1 mentions) Antifade mounting medium, by Vector Laboratories (1 mentions) Anti irs2, by Proteintech (1 mentions) Anti ccnd1, by Wuhan Servicebio Technology (1 mentions) Anti mono adp ribose binding reagent, by Merck Group (1 mentions)

Spelling variants of this product

Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L) secondary antibodies (5 citations) Cy3-conjugated anti-rabbit secondary antibody (3 citations)

14 protocols using cy3 conjugated secondary antibody

Immunofluorescence Staining for Glioma Stem Cells

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Immunofluorescence staining was performed following a standard protocol. Briefly, adhered cells were fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. Subsequently, the samples were permeabilized in 0.5% Triton X-100 and blocked in 5% blocking buffer for 1 h. The cells were then incubated at 4 °C overnight with specific primary antibody (anti-vimentin, anti-FZD7). After washing with PBS three times, the cells were incubated with fluorescence-conjugated secondary antibody (Proteintech) for 1 h and stained with DAPI (4′ 6-diamidino-2-phenylindole) for 10 min, and viewed under a fluorescence microscope.
For GSC identification, tumor spheres were placed on poly-L-ornithine (BD Biosciences)-coated glass coverslips, incubated with anti-CD133 (1:100, Proteintech), and stained with Cy3-conjugated secondary antibody (Proteintech). For the differentiation assay, tumor spheres were seeded in a 24-well plate and cultured in medium supplemented with 10% FBS for 5 days. Differentiated cells were incubated with anti-GFAP (glial fibrillary acidic protein) antibody (1:100, Proteintech), stained with Cy3-conjugated secondary antibody (Proteintech), and counterstained with DAPI.

Liu Q., Guan Y., Li Z., Wang Y., Liu Y., Cui R, & Wang Y. (2019). miR-504 suppresses mesenchymal phenotype of glioblastoma by directly targeting the FZD7-mediated Wnt–β-catenin pathway. Journal of Experimental & Clinical Cancer Research : CR, 38, 358.

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Immunofluorescent Staining of Cardiac Proteins

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Immunofluorescent staining was performed as previously described 18. The primary antibodies included rabbit polyclonal anti‐troponin I (1:50, Santa Cruz, sc‐133117), mouse monoclonal anti‐Sestrin 1 (1:50, Santa Cruz, sc‐376170) and rabbit monoclonal anti‐AMPK (1:50, Cell Signaling Technology, #2532), and the immune complexes were detected with Cy3‐conjugated secondary antibodies (1:100, Proteintech Group, Chicago, IL, USA) or FITC‐conjugated secondary antibodies (1:100, Proteintech Group). The nuclei were stained with DAPI (4′,6′‐diamidino‐2‐phenylindole, 0.5 mg/ml, Sigma‐Aldrich, D9542). The images were captured at 400× using an inverted microscope (CKX41; Olympus Corporation, Tokyo, Japan) or at 630× with a confocal microscope (LSM‐710; Carl Zeiss Inc., Oberkochen, Germany).

Xue R., Zeng J., Chen Y., Chen C., Tan W., Zhao J., Dong B., Sun Y., Dong Y, & Liu C. (2017). Sestrin 1 ameliorates cardiac hypertrophy via autophagy activation. Journal of Cellular and Molecular Medicine, 21(6), 1193-1205.

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Immunohistochemical Analysis of Ki67

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Cells were grouped for different purposes. Fixed the cells with 4% paraformaldehyde at room temperature and blocked with primary antibody block buffer. Using primary antibody anti-Ki67 (1: 50, Proteintech) to incubate the cells at 4 ℃ overnight subsequently. After being washed with PBS, Cy3-conjugated secondary antibodies (1: 200, Proteintech) and Hoechst (Sigma-Aldrich Co., USA) were added at room temperature for cell staining. Finally, observed and photographed using the fluorescence microscope.

Duan B., Shi S., Yue H., You B., Shan Y., Zhu Z., Bao L, & You Y. (2019). Exosomal miR-17-5p promotes angiogenesis in nasopharyngeal carcinoma via targeting BAMBI. Journal of Cancer, 10(26), 6681-6692.

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Immunofluorescence Staining of Troponin I

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Immunofluorescence staining was performed as previously described [12 (link)]. Mouse polyclonal anti-troponin I (1:50, Santa Cruz) was used as the primary antibody, and the immune complexes were detected using Cy3-conjugated secondary antibodies (1:100, Protein-tech Group). The nuclei were stained with DAPI (0.5 mg/ml, Sigma). The images were obtained at 600× using a Nikon A1+ confocal microscope.

Sun Y., Li Y., Liu C., Xue R., Dong B., Huang H., Peng L., Liu J, & Dong Y. (2019). The role of angiopoietin-like protein 4 in phenylephrine-induced cardiomyocyte hypertrophy. Bioscience Reports, 39(7), BSR20171358.

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Hepatic Macrophage Immunofluorescence Staining

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After antigen-retrieval, liver tissue paraffin sections were blocked with 5% goat serum to reduce non-specific binding, then incubated with CD68 (1:200, Proteintech, China) primary antibody overnight at 4 °C. Then, the sections were incubated with the Cy3-conjugated secondary antibodies (1:500, Proteintech, China). The nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, Bioss, China). Images were obtained with a fluorescence microscope.

Liu C., Peng Q., Wei L., Li Z., Zhang X., Wu Y., Wang J., Zheng X., Wen Y., Zheng R., Yan Q., Ye Q, & Ma J. (2022). Deficiency of Lactoferrin aggravates lipopolysaccharide-induced acute inflammation via recruitment macrophage in mice. Biometals, 36(3), 549-562.

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Immunofluorescence Staining for Tumor Tissue Analysis

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Immunofluorescence assay was performed as previous described 42 . Briefly, tumor tissue was fixed in 4% paraformaldehyde for 24 h following by dehydration with 30% sucrose and frozen in O.C.T. Compound (Fisher Scientific, USA), cryosectioned at 8 μm, and mounted on slides. Tissue sections were permeabilized with PBS containing 0.3% Triton X-100 and blocked in PBS containing 1% BSA at 4 °C for 1 h, and then incubated with primary antibodies in PBS at 4 °C overnight, followed by three washings in PBS and further incubation with Cy3-conjugated secondary antibodies (1:300, Proteintech, China) at room temperature for 1 h and by another three washings. The primary antibodies used were anti-IRS2 (1:100, Proteintech, China), anti-CCND1 (1:100, Servicebio, China), anti-Ki67 (1:500, Proteintech, China). Sections were stained with DAPI and mounted using antifade mounting medium (VECTASHIELD, USA), photographed with a fluorescence microscope.

Peng H., Zhang W., Dong H., Yuan J., Li Y., Li F., Yu D., Guan Y, & Zhang F. (2022). CircFAT1 Promotes Lung Adenocarcinoma Progression by Sequestering miR-7 from Repressing IRS2-ERK-mediated CCND1 Expression. International Journal of Biological Sciences, 18(10), 3944-3960.

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Immunofluorescent Analysis of β-Catenin and ADP-Ribose

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Cells (1×10⁵/ml) were fixed in 4% paraformaldehyde for 20 min at room temperature and permeabilized using 0.1% Triton X-100 for 20 min. Samples were blocked in goat serum (Wuhan Boster Biological Technology, Ltd.; cat. no. AR009) for 30 min and subsequently incubated with the following primary antibodies: Anti-β-catenin (Wanleibio Co., Ltd.; cat. no. WL0962a; 1:200) and anti-mono-ADP-ribose binding reagent (EMD Millipore; cat. no. MABE1076; 1:200) at 4°C overnight. The following day, cells were washed in PBS and incubated with Cy3-conjugated secondary antibody (ProteinTech Group, Inc.; cat. no. SA00009-2; 1:200) in the dark for 1 h at room temperature. DAPI (Wuhan Boster Biological Technology, Ltd.) was used for nuclear staining (5 min at room temperature). The images were captured using ZOETM Fluorescent Cell Imager (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ software (version 1.48; National Institutes of Health) The experiment was repeated three times.

Zhang N.N., Lin T., Xiao M., Li Q.S., Li X., Yang L., Wang C.L, & Wang Y.L. (2020). Transcriptome sequencing analysis of mono-ADP-ribosylation in colorectal cancer cells. Oncology Reports, 43(5), 1413-1428.

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Immunofluorescence and Flow Cytometry Analysis

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For immunofluorescence staining, the cells were stained with primary antibody for 1 h at 4 °C, and then stained with fluorescein isothiocyanate (FITC)- or Cy3-conjugated secondary antibody (Proteintech, Chicago, IL, USA) and DAPI (Beyotime, Beijing, China). For the flow cytometry, the expression of CAR was examined by staining with a FITC-labeled recombinant protein of B7-H3 extracellular domain, and the BV510-labeled antiCD4 antibody (Biolegend, San Diego, CA, USA, 357420) and BV650-labled antiCD8 antibody (Biolegend, 344730) were used to determine the expression and ratio of CD8:CD4. Flow cytometry analysis was performed on a BD Fortessa flow cytometer and analyzed using FlowJo 10.6.0 software (BD, Franklin Lakes, NJ, USA).

Tang M., Chen C., Wang G., Wang Y., Zhang Z., Li H., Lu Q., Wang Z., Zhao S., Yang C., Zhong K., Zhang R., Guo L., Yuan Z., Nie C, & Tong A. (2022). Evaluation of B7-H3 Targeted Immunotherapy in a 3D Organoid Model of Craniopharyngioma. Biomolecules, 12(12), 1744.

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Immunofluorescence and Western Blot Analysis of B7-H3 in GBM

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GBM cells were incubated in 24-well plates with poly-L-lysine-treated coverslips at 37°C. After 24 h, specimens were stained with primary antibody or scFv-Fc for 1 h at 4°C and fixed in 4% paraformaldehyde in PBS for 15 min. The slides were stained in turn by a Cy3-conjugated secondary antibody (Proteintech) and DAPI (Beyotime). The immunofluorescent staining was imaged by confocal microscopy.
Western blots were used to probe for B7-H3 expression in isolated glioma primary cells and GBM cell lines. Cells were lysed in radioimmunoprecipitation (RIPA) buffer supplemented with protease inhibitor cocktail (Sigma) for 15 min and then centrifuged at 10,000 × g for 15 min at 4°C. Protein concentrations were determined with a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein were loaded on 10% SDS-PAGE gel and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking in 5% skimmed milk, the membranes were incubated with anti-B7-H3 (1:500, Santa Cruz, F-11) or anti-GAPDH (1:1,000; Beyotime, AF0006) primary antibody overnight. Horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000; Beyotime, A0216) was used at a dilution of 1:2,000. Western blot results were visualized using ChemiScope 6000 Touch (Clinx).

Tang X., Zhao S., Zhang Y., Wang Y., Zhang Z., Yang M., Zhu Y., Zhang G., Guo G., Tong A, & Zhou L. (2019). B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma. Molecular Therapy Oncolytics, 14, 279-287.

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Immunofluorescence Analysis of TET1 Expression

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Cells were fixed in 4% paraformaldehyde at room temperature for 30 min and then permeabilized using 0.5% Triton X-100. Then, cells were washed with cold phosphate-buffered saline to remove free Triton X-100. Subsequently, samples were incubated with primary antibody TET1 at 4 °C overnight. Cells were washed and incubated with Cy3-conjugated secondary antibody (Proteintech, SA00009-1) for 1 h and counterstained with 4′,6-diamidino-2-phenylindole (for nuclear staining). Immunofluorescence was assessed under ZOETM Fluorescent Cell Imager (Bio-Rad) using ImageJ software (version 1.8.0; National Institutes of Health, USA). The assay was repeated in triplicate.

Li M., Tang Y., Li Q., Xiao M., Yang Y, & Wang Y. (2019). Mono-ADP-ribosylation of H3R117 traps 5mC hydroxylase TET1 to impair demethylation of tumor suppressor gene TFPI2. Oncogene, 38(18), 3488-3503.

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