Cy5 conjugated secondary antibody

Manufactured by Thermo Fisher Scientific

Cy5-conjugated secondary antibodies are fluorescent-labeled antibodies used in various immunoassay techniques. They are designed to bind to primary antibodies, allowing for the detection and visualization of target molecules in samples.

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Lab products found in correlation

Tcs sp5 confocal microscope, by Leica (2 mentions) Pecam 1, by BD (1 mentions) Prox1, by R&D Systems (1 mentions) Lyve 1, by Abcam (1 mentions) Biozero bz x700 microscope, by Keyence (1 mentions) Blocking reagent, by PerkinElmer (1 mentions) Lipofectamine ltx plus, by Thermo Fisher Scientific (1 mentions) Neurobasal medium, by Thermo Fisher Scientific (1 mentions) Rabbit anti rfp, by Rockland Immunochemicals (1 mentions) Chicken anti gfp, by Thermo Fisher Scientific (1 mentions) Poly d lysine, by Merck Group (1 mentions) Mouse anti map2, by BD (1 mentions) Anti tuj1, by BioLegend (1 mentions) Cyanine3 (cy3), by Thermo Fisher Scientific (1 mentions) Alexa 488, by Thermo Fisher Scientific (1 mentions) Biotinylated lectin, by Vector Laboratories (1 mentions) Tb0198, by Sangon (1 mentions) Paraformaldehyde (pfa), by Dingguo (1 mentions) Imagexpress micro, by Molecular Devices (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)

Close spelling variants of this product

Cy5-conjugated anti-mouse secondary antibody (4 citations)

7 protocols using cy5 conjugated secondary antibody

Immunofluorescence Analysis of Mouse Skin

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After mouse embryos were fixed with 4% paraformaldehyde (PFA)/PBS at 4 °C and subsequently dehydrated in methanol, the dorsal skin were dissected, rehydrated in PBST (0.2% Tween-20/PBS), and incubated in the blocking buffer consisting of 0.1 M Tris-HCl, pH7.5, 0.5% blocking reagent (PerkinElmer Life Sciences) and 0.15 M NaCl for 1 hour at room temperature (r.t.). The skin samples were incubated with the primary antibodies against LYVE-1 (Abcam), Prox1 (R&D Systems), PECAM-1 (BD Biosciences), or Ki67 (Abcam) at 4 °C overnight and subsequently with Alexa Fluor^®-488-, Alexa Fluor^®-546-, Alexa Fluor^®-594-, Cy3-, and Cy5-conjugated secondary antibodies (Thermo Fisher Scientific). Immunofluorescence images were obtained with Biozero BZ-X700 microscope (Keyence, Japan) or Leica TCS SP5 confocal microscope. The branch number, total vessel length, and width of lymphatic vessels were analyzed by NIH ImageJ software.

Lin Y.C., Ohbayashi N., Hongu T., Katagiri N., Funakoshi Y., Lee H, & Kanaho Y. (2017). Arf6 in lymphatic endothelial cells regulates lymphangiogenesis by controlling directional cell migration. Scientific Reports, 7, 11431.

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Neuronal Culture and Transfection Techniques

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Hippocampal neuronal cultures were prepared from rat embryos at 18 days gestation as previously described [45 (link)]. Neurons were cultured in Neurobasal medium (Life Technologies) supplemented with B27, L-glutamine, penicillin, and streptomycin. Neurons were plated on to glass coverslips coated with poly-D-lysine (0.1mg/ml, Sigma) in a 37°C incubator with 5 % CO2. Neurons were plated at a density of 105,000 cells/cm² for dendrite branching experiments and at a density of 50,000 cells/cm² for spine analysis. Neurons were transfected with pEGFP-C1, pEGFP-C1-Cypin or pEGFP-C1-CypinS using Lipofectamine LTX+PLUS (Invitrogen) following the manufacturer’s protocol for dendrite branching and using calcium phosphate transfection for spine analysis [45 (link)]. For all conditions, neurons were co-transfected with pGW1-mRFP to visualize dendrites and axon. For dendrite branching experiments, neurons were transfected at 7 days in vitro (DIV7) and fixed with 4% paraformaldehyde (PFA) in PBS at DIV12. For spine analysis, neurons were transfected on DIV14 and fixed on DIV17. Cells were immunostained with chicken anti-GFP (1:250; Thermo Fisher Sci.), rabbit anti-RFP (1:250; Rockland) and mouse anti-MAP2 (1:250; BD PharMingen). Immunostaining was visualized with Cy2-, Cy3-, or Cy5-conjugated secondary antibodies (1:500; Thermo Fisher Sci.).

Patel M.V., Swiatkowski P., Kwon M., Rodriguez A.R., Campagno K, & Firestein B.L. (2018). A novel short isoform of cytosolic PSD-95 interactor (cypin) regulates neuronal development. Molecular neurobiology, 55(8), 6269-6281.

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Immunostaining Protocol for Neural and Liver Cell Markers

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The following primary antibodies were used: Anti-PGP 9.5/UCHL (rabbit, 1:500, Proteintech, Cat# 14730-1-AP), Anti-TUJ1 (mouse, 1:500, BioLegend, Cat# 801201), Anti-Tubulin beta Ⅳ (TUBB4) (rabbit, 1:400, Abcam, Cat# ab179509), Anti-Tubulin beta Ⅳ (TUBB4) (mouse, 1:300, Sigma-Aldrich, Cat# T7941), Anti-CK19 (rat, 1:300, DSHB, Troma-Ⅲ-c), Anti-CYP2A6 (mouse, 1:400, Invitrogen, Cat# PIMA525758), Anti-GGT7 (rabbit, 1:500, Proteintech, Cat# 24674-1-AP). Before immunostaining, tissue sections were rinsed in PBS for 5 min to remove OCT and then placed in 3% hydrogen peroxide-MeOH solution for 10 min to quench intrinsic peroxidase activity. Sections were then puddled with 0.01 M citrate buffer, pH 6.0, and steam heated for 10 min in a commercial food steamer for antigen retrieval. Sections were rinsed in PBS and then blocked with 10% donkey serum 5% nonfat dry milk/4% bovine serum albumin/0.1% Triton X-100 in PBS for 10 min and incubated overnight with primary antibody at 4°C in a humid chamber. The following day, the staining was visualized by incubating with Alexa 488, Cy3, or Cy5-conjugated secondary antibodies (Invitrogen, Carlsbad, CA; Jackson ImmunoResearch Laboratories, West Grove, PA) at 1:200 dilution. Immunofluorescent-stained sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei and coverslipped with glycerol/n-propyl gallate mounting medium.

Nishijima H., Zunitch M.J., Yoshida M., Kondo K., Yamasoba T., Schwob J.E, & Holbrook E.H. (2022). Rapid fluorescent vital imaging of olfactory epithelium. iScience, 25(5), 104222.

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Glycan Microarray Binding Analysis

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For interrogation of the printed subarrays, GBPs were diluted in binding buffer (TSM buffer with 1% BSA and 0.05% Tween20). The array was interrogated with the following biotinylated lectins (Vector Labs): Helix pomatia agglutinin (HPA), Maakia amurensis lectin (MAL), Sambucus nigra agglutinin (SNA), and soybean agglutinin (SBA), which are specific for defined GAEAB or glycophage structures on the array. An aliquot (200 μl) of each GBP or antibody was applied to individual subarrays and incubated for 1 h at room temperature. After washing with TSM buffer to remove excess GBP, biotinylated lectins were detected by a second incubation with 0.5 μg/ml Cy5-streptavidin (Cy5-SA; Invitrogen Life Technologies), anti-glycan antibodies were detected by a secondary incubation with 5 μg/ml Cy5-conjugated secondary antibodies (Invitrogen Life Technologies) at room temperature for 1 h followed by a wash step to remove excess reagent and finally with water to remove residual salt.

Çelik E., Ollis A.A., Lasanajak Y., Fisher A.C., Gür G., Smith D.F, & DeLisa M.P. (2014). Glycoarrays with engineered phages displaying structurally diverse oligosaccharides enable high-throughput detection of glycan-protein interactions. Biotechnology journal, 10(1), 199-209.

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Immunofluorescence Staining of Cells

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Cells were fixed with 4% paraformaldehyde (Dingguo Changsheng Biotechnology) at 37°C for 30 min, then stained with the lectin diluent buffer containing 2 μL WGA (5 mg/mL) at 4°C for 30 min. After discarding the staining solution, cells were permeabilized with 0.2% Triton X-100 (Shanghai Sangon Biotech, TB0198) at 4°C for 10 min and blocked by goat serum at room temperature for 2 h. Cells were then incubated with the anti-Biotin antibody overnight at 4°C, washed with 0.05% Triton X-100, and stained with CY5-conjugated secondary antibodies (Invitrogen) for 1 h at room temperature. Cell images were captured with TCS SP5 Leica confocal microscope.

Luo Q., Liu Q., Cheng H., Wang J., Zhao T., Zhang J., Mu C., Meng Y., Chen L., Zhou C., Lei H., Yang J., Chen G., Li Y., Pan L., Chen Q, & Zhu Y. (2022). Nondegradable ubiquitinated ATG9A organizes Golgi integrity and dynamics upon stresses. Cell reports, 40(7).

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Quantifying Dopaminergic Differentiation in SNCA-GFP SH-SY5Y Cells

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To examine neuronal differentiation of SNCA-GFP SH-SY5Y cells, phorbol ester 12-o-tetradecanoylphorbol-13-acetate (TPA) (120 nM; Enzo Life Sciences, Farmingdale, NY, USA) was used to promote DAergic differentiation [27 (link)]. TPA-treated SNCA-GFP-expressing SH-SY5Y cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, blocked in 2% bovine serum albumin (BSA), and stained with primary anti-tyrosine hydroxylase (TH) antibody (1:1000; Millipore #612300) at 4 °C overnight, followed by Cy5-conjugated secondary antibody (Invitrogen) at room temperature for 2 h. Nuclei were detected using 4′-6-diamidino-2-phenylindole (DAPI; 0.1 g/mL; Sigma-Aldrich). DAergic differentiation was examined by simultaneous fluorescent imaging of GFP (482 ± 17.5 nm excitation and 536 ± 20 nm emission) and Cy5 (628 ± 20 nm excitation and 692 ± 20 nm emission) fluorescence using a high content analysis (HCA) system (ImageXpressMICRO; Molecular Devices, San Jose, CA, USA). For quantification of TH-positive cells, 5 × 10⁴ SNCA-GFP expressing cells (green) from three independent experiments were analyzed for TH expression (red).

Chen C.M., Lin C.H., Wu Y.R., Yen C.Y., Huang Y.T., Lin J.L., Lin C.Y., Chen W.L., Chao C.Y., Lee-Chen G.J., Su M.T, & Chang K.H. (2020). Lactulose and Melibiose Inhibit α-Synuclein Aggregation and Up-Regulate Autophagy to Reduce Neuronal Vulnerability. Cells, 9(5), 1230.

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Subcellular Protein Detection by Western Blot

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An equivalent amount of each subcellular fraction was mixed with 2x Laemmli sample buffer and heated in a boiling water bath for 10 minutes prior to loading cell equivalents on a 10% SDS-PAGE gel. Gels were run at 125 V until the dye front reached the bottom of the gel and then proteins were transferred to a nitrocellulose membrane at 25 V for 1.5 hours. Membranes were blocked with NAP-blocker (G-Biosciences) diluted 1:2 with Tris-buffered saline containing 0.1% Tween-20 (TBST) and then cut to allow for simultaneous probing with each antibody. Primary antibodies against Ply at 1:1000 (Statens Serum Institut) and CodY at 1:1500 (a gift from A.L. Sonenshein) were diluted accordingly in NAP-blocker mixed 1:4 with TBST and applied to each membrane at room temperature for 1 hour with rocking. Membranes were washed three times for 5 minutes each with TBST. Appropriate Cy5-conjugated secondary antibody at 1:1000 (Invitrogen, Inc.) was applied to each membrane as described above for the primary and then each blot was washed as described above. Membranes were scanned with a Fuji FLA-9000 instrument and the amount of fluorescence was quantitated using MultiGauge analysis software (Fujifilm, Corp.)

Greene N.G., Narciso A.R., Filipe S.R, & Camilli A. (2015). Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release. PLoS Pathogens, 11(6), e1004996.

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