Cy3 anti mouse secondary antibody

Manufactured by Jackson ImmunoResearch

Sourced in United Kingdom

Cy3 anti-mouse secondary antibody is a fluorescently labeled antibody that binds to mouse primary antibodies. It is used in immunoassays to detect and visualize target proteins or antigens recognized by mouse antibodies.

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Lab products found in correlation

Lsm 780, by Zeiss (2 mentions) Triton x 100, by Merck Group (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions) Tcs sp8 microscope, by Leica (2 mentions) H 1000, by Vector Laboratories (2 mentions) Secondary antibody incubation, by Jackson ImmunoResearch (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Vectashield containing 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions) Dmi3000b fluorescence microscope, by Leica (1 mentions) Dic n2 plan apo, by Nikon (1 mentions) Coolsnap photometrics camera, by Nikon (1 mentions) Eclipse 90i, by Nikon (1 mentions) α sma primary antibody, by Santa Cruz Biotechnology (1 mentions) Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions) Dm3000 microscope, by Leica (1 mentions) Lsm 800, by Zeiss (1 mentions) Fluorescent microscope, by Zeiss (1 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions) Vectorshield dapi, by Vector Laboratories (1 mentions) Anti mouse cy5, by Jackson ImmunoResearch (1 mentions)

12 protocols using cy3 anti mouse secondary antibody

Immunofluorescence Staining of Key Signaling Proteins

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For PI (3,4,5)P3, PTEN, USP11, or FOXO1 immunofluorescence staining, cells were incubated with anti-PI(3,4,5)P3 antibody, anti-PTEN, anti-USP11, or anti-FOXO1. Cells were then washed and incubated with Cy3 anti-mouse secondary antibody (1:5000; Jackson ImmunoResearch) for 1 h. Slides were then mounted in Vectashield containing 4′,6-diamidino-2-phenylindole (Vector Lab). Immunofluorescence images were generated using a Leica DMI 3000B fluorescence microscope (Leica).

Park M.K., Yao Y., Xia W., Setijono S.R., Kim J.H., Vila I.K., Chiu H.H., Wu Y., Billalabeitia E.G., Lee M.G., Kalb R.G., Hung M.C., Pandolfi P.P., Song S.J, & Song M.S. (2019). PTEN self-regulates through USP11 via the PI3K-FOXO pathway to stabilize tumor suppression. Nature Communications, 10, 636.

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Immunofluorescence Assay for PML Protein

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Cells (5 × 10⁶/well in 12-well plates) were seeded in 1 mL RPMI-1640 supplemented with 5% FBS overnight and washed with 1x PBS twice on next day. Cells were cultured with 0.5 mL serum-free RPMI for another 24 h Ovcar3 cells were then treated 1 μM KU60019 and 10 μM fenofibrate; Ovcar10 cells were treated 10 μM KU60019 and 25 μM fenofibrate in RPMI + 0.1% FBS for 3 days. KU60019 was administered 5 h prior to fenofibrate. Cells were fixed in 4% paraformaldehyde for 10min and permeabilized in 0.2% Triton X-100 for 5 min. Cells then were stained with PML (1:200, Santa Cruz, Cat# sc-966) in 3% BSA/PBS at room temperature for 1 h. Cells were further incubated with 0.15 μg/ml DAPI in PBS (1 min), mounted, and sealed. Cells were washed three times and then incubated in Cy3 anti-mouse secondary antibody (1:5000, Jackson ImmunoResearch Labs, Cat# 715-165-150) in 3% BSA/PBS at room temperature for 1 h. Finally, cells were incubated with 0.15 μg/ml DAPI in PBS for 1 min, washed three times with PBS, mounted, and sealed. Images were acquired at room temperature using a Nikon Eclipse 90i microscope with a 20x/0.17 objective (Nikon DIC N2 Plan Apo) equipped with a CoolSNAP Photometrics camera.

Chen C.W., Buj R., Dahl E.S., Leon K.E, & Aird K.M. (2020). ATM inhibition synergizes with fenofibrate in high grade serous ovarian cancer cells. Heliyon, 6(9), e05097.

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Immunofluorescence Analysis of αSMA Expression

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Hs68, PCF54 and PCF55 cells were seeded in DMEM-F12 complete medium at a density of 1 × 10⁴ cells per well in 24-well plates on glass coverslips and grown as a monolayer for 24 h. After that, the cells were rinsed with PBS, fixed, and permeabilized with methanol-acetone (1:1) in -20 °C for 20 min, then washed with PBS and blocked with 2% BSA. Cells were incubated with αSMA primary antibody (sc-32,251, Santa Cruz Biotechnology, USA; 1:50 dilution in PBS, 1% BSA) overnight in + 4 °C and with Cy3-anti mouse secondary antibody (115-165-071, Jackson Immunoresearch, UK) for 1 h in room temperature in dark. Next, cells were washed with PBS and mounted on glass slides in ProLong™ Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, USA) and incubated in + 4˚C, overnight. Fluorescence imaging was done on Leica DM3000 microscope (Leica Microsystems GmbH, Germany).

Sadovska L., Zayakin P., Bajo-Santos C., Endzeliņš E., Auders J., Keiša L., Jansons J., Lietuvietis V, & Linē A. (2022). Effects of urinary extracellular vesicles from prostate cancer patients on the transcriptomes of cancer-associated and normal fibroblasts. BMC Cancer, 22, 1055.

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Immunofluorescent Staining of Vascular Cells

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Before immunostaining, cells were permeabilized for 2 min at room temperature in 0.5% Triton X-100 (Sigma-Aldrich) in 4% paraformaldehyde solution. Cells were then washed three times with PBS, incubated in 4% paraformaldehyde solution for 20 min at room temperature, and then probed with fluorophore-tagged antibodies. Samples were incubated with antibodies targeting VE-cadherin (Cayman Chemical, rabbit; 1:100) and smooth muscle α-actin (Santa Cruz Biotechnology, mouse; 1:25) prepared in blocking buffer (0.1% bovine serum albumin in universal buffer) for 1 hour in 37°C. Samples were then incubated in secondary antibody (Alexa Fluor 488 anti-rabbit secondary antibody or Cy3 anti-mouse secondary antibody, Jackson ImmunoResearch; 1:100 dilution) for 1 hour in 37°C. A Zeiss LSM 780 or a Zeiss LSM 800 laser scanning confocal microscope was used for confocal imaging. Cell viability was measured using calcein-AM and ethidium homodimer-1 staining and imaged with a Zeiss LSM 780.

Jin Q., Bhatta A., Pagaduan J.V., Chen X., West-Foyle H., Liu J., Hou A., Berkowitz D., Kuo S.C., Askin F.B., Nguyen T.D., Gracias D.H, & Romer L.H. (2020). Biomimetic human small muscular pulmonary arteries. Science Advances, 6(13), eaaz2598.

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Vinculin Immunofluorescence Staining

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Cells were then washed with warm 1×
phosphate-buffered saline (PBS) twice before being fixed with 4% formaldehyde
at room temperature for 15 min followed by permeabilization with a
solution of 0.1% Triton X-100 in PBS at room temperature for 5 min.
To block nonspecific binding, the samples were incubated in 1% bovine
serum albumin (BSA) in PBS for 1 h at room temperature. After blocking,
an anti-vinculin mouse primary antibody at a dilution of 1:500 in
1% BSA in PBS was added to the samples and incubated for 1 h at room
temperature. The samples were consequently washed with 0.5% Tween
20 in PBS three times for 5 min. A Cy3 anti-mouse secondary antibody
(1:200) (Jackson ImmunoResearch Laboratories Inc.), Phalloidin Alexa
Fluor 488 (1:100) (Thermofisher Scientific, U.K.) in 1% BSA in PBS,
was consequently added to the samples and incubated for 1 h at room
temperature followed by another three 5 min washes with 0.5% Tween
20 in PBS. The nuclei of the cells were stained using Vectorshield-DAPI
(Vector Laboratories) before being imaged with a fluorescent microscope
(Zeiss, GmbH, Germany). All reagents unless otherwise stated were
sourced from Sigma-Aldrich, U.K.

Sarrigiannidis S.O., Moussa H., Dobre O., Dalby M.J., Tamimi F, & Salmeron-Sanchez M. (2020). Chiral Tartaric Acid Improves Fracture Toughness of Bioactive Brushite–Collagen Bone Cements. ACS Applied Bio Materials, 3(8), 5056-5066.

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Immunostaining of Embryonic Samples

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One-day-old adults were incubated in 1x M9 solution for 4–5 hours and their embryos were removed, fixed, and stained^{77 (link)}. Embryos were attached to microscope slides coated with poly-lysine and containing Teflon spacers. Slides were frozen on dry ice, embryos were permeabilized by freeze-crack method and submerged in 100% MeOH for 5–10 minutes at - 20°C. Embryos were submerged for 5 minutes twice in PBS, then once in PBT (PBS plus 0.1% Tween). Embryos were incubated in primary antibody overnight at 4°C. Embryos were then washed in PBT for 5 minutes three times and then incubated in secondary antibody for 1 hour at 37°C. Embryos were washed once in PBT then twice in PBS, mounted in Vectashield (Vector Laboratories), and stored at 4°C. The following primary antibodies were used: anti-HA primary antibody (Abcam, 1:200), anti-Myc (Abcam, 1:100), anti-GFP (Abcam, 1:200), anti-PAR-3 (DSHB), anti-GIP-1 (GenScript^{7 (link)}). The following secondary antibodies were used: CY3-anti-mouse secondary antibody (Jackson Immunoresearch Laboratories, 1:200), Streptavidin Alexa Fluor 488 (Invitrogen, 1:200), DAPI (Sigma, 1:10,000), 647-anti-rabbit (Jackson Immunoresearch Laboratories, 1:50), 488-anti-mouse (Jackson Immunoresearch Laboratories, 1:200), 488 anti-rabbit (Jackson Immunoresearch Laboratories, 1:200), Cy5 anti-mouse (Jackson Immunoresearch Laboratories, 1:50).

Sanchez A.D., Branon T.C., Cote L.E., Papagiannakis A., Liang X., Pickett M.A., Shen K., Jacobs-Wagner C., Ting A.Y, & Feldman J.L. (2021). Tissue-specific proximity labeling reveals non-centrosomal microtubule-organizing center components required for microtubule growth and localization. Current biology : CB, 31(16), 3586-3600.e11.

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Immunostaining of Drosophila Brain

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Flies were collected and brains dissected in phosphate-buffered saline (PBS), and then fixed in 4% PFA at RT for 30 mins, blocked with 5% normal goat serum (NGS) in PBST (0.3% Trition X-100 in PBS) at RT for 1 hr. Samples were incubated with primary at 4C overnight, and washed 3 3 15 min in PBST. Secondary antibody incubation (Jackson ImmunoResearch Inc.) was performed at R.T. for 1 hour, then washed 3 3 15 min with PBST and mounted with Vectashield Mounting Medium (H-1000, Vector Laboratories). Imaging was performed using a Leica TCS SP8 microscope. Anti-Fasciclin II was used at 1:20 (1D4, Developmental Studies Hybridoma Bank). Anti-mouse Cy3 secondary antibody was used at 1:500 (Cat# 515-165-003, Jackson ImmunoResearch).

Zhang Z., So K., Peterson R., Bauer M., Ng H., Zhang Y., Kim J.H., Kidd T, & Miura P. (2019). Elav-Mediated Exon Skipping and Alternative Polyadenylation of the Dscam1 Gene Are Required for Axon Outgrowth. Cell reports, 27(13), 3808-3817.e7.

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Immunostaining of Drosophila Brain

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Quantifying Muscle Stem Cell Proliferation

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Six days prior to harvesting, mice were given the thymidine analogue 5-Bromo-2′-deoxyuridine (BrdU, 1 mg/ml, Sigma, B5002) in the drinking water supplemented with sucrose (25 mg/ml). Muscle stem cells were isolated by FACS, plated on µ-Slide 8 Well (Ibidi, 80826) in medium (40% DMEM, 40% MCDB (Sigma), 20% FBS, 2% Ultroser (Pall), 1% Penicillin-Streptomycin (ThermoFisher)) at 37 °C 5% CO₂ 3% O₂ to allow adherence, fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) for 5 min at RT, washed twice 5 min with PBS, permeabilised in 0.5% Triton X-100 (Sigma) for 5 min at RT, washed three times with PBS, incubated with DNAse I (Roche, 04536282001) at 1 unit/µL in PBS at 37 °C for 30 min, washed three times with PBS, blocked in 10% goat serum (GS, Gibco) for 30 min at RT, incubated with mouse anti-BrdU antibody (1/100, BD, 347580) in 2% GS for 2 h at RT, washed three times 5 min in PBS, incubated with anti-mouse Cy3 secondary antibody (1/500, Jackson ImmunoResearch, 115-165-205) and Hoechst (1 µg/ml) in 2% GS for 45 min at RT, and washed four times with PBS. Images were taken with a Zeiss LSM800 confocal (×40 objective).

Hernando-Herraez I., Evano B., Stubbs T., Commere P.H., Jan Bonder M., Clark S., Andrews S., Tajbakhsh S, & Reik W. (2019). Ageing affects DNA methylation drift and transcriptional cell-to-cell variability in mouse muscle stem cells. Nature Communications, 10, 4361.

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Immunostaining of Cytochrome c in Neurons

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The status of cyt c (whether intact in the mitochondria or released) was examined by immunostaining with cyt c antibodies. Briefly, sympathetic neurons were cultured for 4 days after which they were either left untreated or deprived of NGF for various time points. Neurons and other cell lines were fixed in 4% paraformaldehyde and incubated overnight in anti-cyt c primary antibody (556432, BD Biosciences) followed by a 2 h incubation with anti-mouse Cy3 secondary antibody (Jackson Labs). Nuclei were stained with Hoechst 33258 (Molecular Probes).

Gama V., Swahari V., Schafer J., Kole A.J., Evans A., Huang Y., Cliffe A., Golitz B., Sciaky N., Pei X.H., Xiong Y, & Deshmukh M. (2014). PARC/CUL9 Mediates the Degradation of Mitochondrial-released Cytochrome c and Promotes Survival in Neurons and Cancer Cells. Science signaling, 7(334), ra67.

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