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Cy3 conjugated donkey anti rabbit secondary antibody

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Cy3-conjugated donkey anti-rabbit secondary antibody is a laboratory reagent used in immunological techniques. It is a secondary antibody that binds to rabbit primary antibodies and is conjugated with the fluorescent dye Cy3, which can be used for detection and visualization purposes.

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Lab products found in correlation

Cy3 conjugated donkey anti goat secondary antibody, by Jackson ImmunoResearch (3 mentions) Fitc conjugated donkey anti goat secondary antibody, by Jackson ImmunoResearch (2 mentions) Vectashield mounting medium, by Vector Laboratories (2 mentions) Cytation 5 automated microscope, by Agilent Technologies (1 mentions) Rabbit anti myc antibody, by Cell Signaling Technology (1 mentions) Fitc conjugated donkey anti mouse secondary antibody, by Jackson ImmunoResearch (1 mentions) Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions) Ab53554, by Abcam (1 mentions) Ab955, by Abcam (1 mentions) Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions) Apotome, by Zeiss (1 mentions) Anti phospho h2ax, by Cell Signaling Technology (1 mentions) Anti flag m2, by Merck Group (1 mentions) Anti rad51, by Santa Cruz Biotechnology (1 mentions) Tissue tek oct, by Sakura Finetek (1 mentions) X81 fluorescence microscope, by Olympus (1 mentions) Anti neun primary antibody, by Cell Signaling Technology (1 mentions) Z fix solution, by Anatech (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade mounting medium, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Cy3-conjugated secondary antibody (148 citations) Anti-rabbit Cy3 (101 citations) Anti-rabbit secondary antibody (44 citations) Cy3-conjugated anti-rabbit secondary antibody (26 citations) Donkey anti-rabbit secondary antibody (14 citations) Cy3-conjugated donkey anti-rabbit antibody (9 citations) Cy3-conjugated anti-rabbit antibody (9 citations) Donkey anti-rabbit Cy3 antibody (8 citations) Donkey anti-rabbit Cy3 secondary antibody (6 citations) Anti-rabbit-Cy3 secondary antibody (5 citations)

10 protocols using cy3 conjugated donkey anti rabbit secondary antibody

1

Immunohistochemical Analysis of Kidney Injury Markers

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Paraffin‐embedded kidney sections (4 m) were deparaffinized in xylene and rehydrated in graded alcohol concentrations. Sections were subsequently boiled with 10 mM sodium citrate in a water bath for 30 min. The sections were then blocked in 3% normal donkey serum (diluted in PBS containing 0.1% Triton‐X) for 1 hr at 4°C, and then incubated overnight at 4°C with diluted primary antibodies as follows: goat anti‐NEP (1:500, Cat # AF1126, R&D Systems) and rabbit anti‐ACE2 (1:500, Cat # HPA000288, Sigma). Then, the sections were incubated with CY3 conjugated donkey anti‐goat secondary antibody (1:500, code # 705‐165‐003, Jackson Immunoresearch). For the co‐localization, FITC conjugated donkey anti‐goat secondary antibody (1:500, code # 705‐095‐147, Jackson Immunoresearch) and CY3 conjugated donkey anti‐rabbit secondary antibody (1:500, code # 711‐165‐152, Jackson Immunoresearch) were used and incubated for 2 hr at 4°C. Sections were mounted using a vectashield‐mounting medium (Vector). Images were acquired using a fluorescence microscope (Optronics).
For morphological evaluation of kidney injury, sections were stained with periodic acid‐Schiff (PAS) by AML laboratories. Sections were examined under bright color field and images were acquired using a 40× objective on the Cytation 5 Automated Microscope (BioTek Instruments Inc.).
Alawi L.F., Emberesh S.E., Owuor B.A., Chodavarapu H., Fadnavis R., El‐Amouri S.S, & Elased K.M. (2020). Effect of hyperglycemia and rosiglitazone on renal and urinary neprilysin in db/db diabetic mice. Physiological Reports, 8(3), e14364.
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2

Histological Assessment of Spinal Cord Injury

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Mice were sacrificed one day after T4 and T10 SCI (n = 2 each) for histological assessment of the lesioned spinal cord. Luxol fast blue and hematoxylin and eosin staining methodologies which examine myelin and general structure of the injured spinal cord were undertaken as we previously described [32 (link)]. We also performed fluorescent histology to assess GFAP expression in the lesioned spinal cord dorsal horn 1 day after T4 and T10, using routinely used methodologies, e.g., [8 (link)]. For fluorescent histology, the spinal cord sections were blocked in 5% donkey serum, then incubated in GFAP antibody raised in rabbit (1:1000, # NB300-141) overnight. They were then incubated in Cy3- conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch Lab #711–165-152) for 1 h. Additional details on these methodologies and the results are provided as supplemental material.
Parvin S., Williams C.R., Jarrett S.A, & Garraway S.M. (2021). Spinal Cord Injury Increases Pro-inflammatory Cytokine Expression in Kidney at Acute and Sub-chronic Stages. Inflammation, 44(6), 2346-2361.
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3

Visualizing Surface FasII Expression in S2R+ Cells

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S2R+ cells transfected with the pAc-Myc-fasII construct with or without mical-like dsRNA were incubated for 72 h and then treated with 100 μg/ml cycloheximide for 5 h to inhibit protein synthesis. To visualize surface FasII molecules, the cells were incubated with rabbit anti-Myc antibody (Cell Signaling, USA; 1:100) in culture medium for 1 h at 4°C and then fixed for 20 min with 4% formaldehyde in PBS. After washing three times with PBS, cells were incubated with Cy3-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, USA; 1:200) for 30 min. To visualize the total pool of FasII, the same cells were then permeabilized with 0.2% Triton X-100 in PBS for 10 min and sequentially incubated with mouse anti-FasII antibody (DSHB, USA; 1:50) for 1 h and FITC-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, USA; 1:200) for 30 min. S2R+ cells showed no detectable levels of endogenous FasII based on anti-FasII immunostaining. Images of immunostained cells were acquired using a LSM 700 laser-scanning confocal microscope (Carl Zeiss, Germany). Images were processed using the ZEN imaging software. To quantify surface FasII expression, the fluorescence intensity of surface Myc-FasII (red) was normalized to the total Myc-FasII (green) fluorescence intensity.
Nahm M., Park S., Lee J, & Lee S. (2016). MICAL-like Regulates Fasciclin II Membrane Cycling and Synaptic Development. Molecules and Cells, 39(10), 762-767.
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4

Immunohistochemical Analysis of Rat Brain

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Brain cryosections with a thickness of 8 μm were obtained using a Ultrapro 5000 cryostat (Vibratome). All the sections were fixed with Z-fix solution for 15 min, and then the non-specific binding sites were blocked by PBS containing 1% bovine serum albumin (BSA) for 30 min. Slices were incubated with primary antibodies at 4 °C overnight and then with secondary antibodies at room temperature for another 60 min. After each step, slices were washed gently 3 times with PBS containing 0.05 % tween 20 (PBST) for 5 min. For different staining targets, the antibodies and concentrations were as follows: rabbit anti-rat PBR (TSPO) antibody (1:50; Santa Cruz; sc-20120), mouse anti-rat CD68 antibody (1:100; Abcam; ab955) and goat anti-rat GFAP antibody (1:100; Abcam; ab53554); Cy3-conjugated donkey anti-rabbit secondary antibody (1:200; Jackson ImmunoResearch Laboratories), Dylight 488-conjugated donkey anti-rabbit secondary antibody (1:200; Jackson ImmunoResearch Laboratories) and Cy3-conjugated donkey anti-goat secondary antibody (1:200, Jackson ImmunoResearch Laboratories). All tissue slices were mounted with medium containing 4′, 6-diamidino-2-phenylindole (DAPI), and then observed by an epifluorescence microscope (X81; Olympus).
Wang Y., Yue X., Kiesewetter D.O., Niu G., Teng G, & Chen X. (2014). PET imaging of neuroinflammation in a rat traumatic brain injury model with radiolabeled TSPO ligand DPA-714. European journal of nuclear medicine and molecular imaging, 41(7), 1440-1449.
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5

Quantifying DNA Damage and Repair Foci

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For imaging γ-H2AX and Rad51
foci, the cells growing on the coverslips were treated as indicated.
Then the cells were fixed with 3.7% formaldehyde and permeabilized
with 0.5% triton X-100. The cells were washed with 1× PBS and
blocked with 3% BSA before incubating with primary antibodies. The
following primary antibodies were used: anti-phospho-H2AX (rabbit,
Cell Signaling Technology, catalogue no. 9718, 1:400); anti-Rad51
(rabbit, Santa Cruz Biotechnology, catalogue no. sc-8349, 1:100);
anti-LA (mouse, Sigma, catalogue no. SAB4200236, 1:1000); anti-FLAG
M2 (mouse, Sigma, catalogue no. F3165, 1:400). The cells were then
incubated with secondary antibodies for 1 h at room temperature. Cy-3-conjugated
donkey antirabbit secondary antibody (Jackson Immunoresearch) was
used at 1:1000 dilution. Coverslips were mounted in ProLong Gold AntiFade
reagent with DAPI (Life Technologies), and images were acquired with
a fluorescence microscope ApoTome (Zeiss). The cells were considered
foci-positive if >10 foci were observed per nucleus. DAPI was used
to count cell nuclei. Around 100 cell nuclei were analyzed for each
experiment, and results are shown as the average of three independent
cell preparations. For colocalization analysis, the Z-stack of images were reconstructed, and colocalization Pearson correlation
coefficients were determined using the Coloc module in the Imaris
software package (Bitplane).
Li B.X., Chen J., Chao B., Zheng Y, & Xiao X. (2018). A Lamin-Binding Ligand Inhibits Homologous Recombination Repair of DNA Double-Strand Breaks. ACS Central Science, 4(9), 1201-1210.
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6

Mature Neuron Labeling in TBI Mouse Model

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At designated time point, mice were perfused with 4% polyformaldehyde (PFA) (20–30 mL) before dissection of brain tissue. The brain tissue was dehydrated with 30% sucrose 24 h before being embedded in Tissue Tek OCT (Sakura Finetek), followed by snap freezing in liquid nitrogen. The OCT embedded tissue blocks were kept at −80 °C for cryosection. The obtained brain slices (15 µm-thick) were fixed in Z-fix solution (Anatech) for 10 min, and permeabilized with 0.1% Triton X-100 for 5 min. To stain the mature neuron cells at the periphery of TBI site, a mature neuron marker, NeuN, was chosen. Anti-NeuN primary antibody was obtained from Cell Signaling Technology (1:150, Cat# 12943). The Cy3-conjugated donkey anti-rabbit secondary antibody (1:200, Cat# 711165-152, Jackson ImmunoResearch) was used. The nuclei were counterstained with DAPI using Vectashield mounting medium (Vector Lab). The images were taken from Olympus X81 fluorescence microscope.
Wang Z., Wang Y., Wang Z., Zhao J., Gutkind J.S., Srivatsan A., Zhang G., Liao H.S., Fu X., Jin A., Tong X., Niu G, & Chen X. (2015). Polymeric Nanovehicle Regulated Spatiotemporal Real-Time Imaging of the Differentiation Dynamics of Transplanted Neural Stem Cells after Traumatic Brain Injury. ACS nano, 9(7), 6683-6695.
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7

Immunofluorescence Analysis of Epithelial Markers

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Immunofluorescence was performed with MUC2, claudin-3 and ZO-1 antibodies. Paraffin sections were deparaffinized and treated with heat-mediated antigen retrieval using sodium citrate buffer (pH 6.0). After three washes with TBS, the sections were incubated with 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature to block non-specific binding. Sections were then incubated with rabbit anti-MUC2 (1:200; H-300, Santa Cruz Biotechnology, sc-15534), rabbit anti-claudin 3 (1:100; Invitrogen, 34-1700) and rabbit anti-ZO-1 (1:250; Invitrogen, 61-7300) overnight at 4°C. The slides were washed three times and incubated with Cy3-conjugated donkey anti-rabbit secondary antibody (1:300; Jackson ImmunoResearch). Samples were counterstained with Hoechst 33342 (Invitrogen) and washed three times with TBS. The slides were mounted with Prolong Gold anti-fade mounting medium (Invitrogen). Nuclei were counterstained with diamidino-2 phenylindole (DAPI) (Sigma-Aldrich, St Louis, MO, USA). The slides were imaged using a fluorescent microscope (Nikon, Japan), and intensity was calculated using ImageJ software.
Singh P., Sanchez-Fernandez L.L., Ramiro-Cortijo D., Ochoa-Allemant P., Perides G., Liu Y., Medina-Morales E., Yakah W., Freedman S.D, & Martin C.R. (2020). Maltodextrin-induced intestinal injury in a neonatal mouse model. Disease Models & Mechanisms, 13(8), dmm044776.
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8

Neprilysin and ACE2 Protein Expression in Rosiglitazone Therapy

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Rosiglitazone was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). Primary polyclonal goat anti‐NEP (Cat # AF1126) and donkey anti‐goat secondary antibody (Cat # HAF017) were purchased from R&D Systems. CY3 conjugated donkey anti‐goat secondary antibody (code # 705‐165‐147), CY3 conjugated donkey anti‐rabbit secondary antibody (code # 711‐165‐152) and FITC conjugated donkey anti‐goat secondary antibody (code # 705‐095‐147) were purchased from Jackson Immunoresearch. Primary polyclonal rabbit anti‐ACE2 form Sigma (Cat # HPA000288). Mouse Albumin ELISA kit was from Bethyl Laboratories (Cat # E90‐134). Mouse Neprilysin DuoSet ELISA development kit from R&D systems (Cat # DY1126). SensoLyte® 520 NEP activity assay kit from AnaSpec EGT group (Cat# 72223).
Alawi L.F., Emberesh S.E., Owuor B.A., Chodavarapu H., Fadnavis R., El‐Amouri S.S, & Elased K.M. (2020). Effect of hyperglycemia and rosiglitazone on renal and urinary neprilysin in db/db diabetic mice. Physiological Reports, 8(3), e14364.
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9

Tracing Neurons Projecting to PVN

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The fluorescent tracer latexgreen (4%, 100 nL, ThermoFisher) was injected bilaterally into the region of the RVLM in three rats 7 days prior to sacrifice. This allowed the tracer enough time to be transported from the RVLM to the neurons that project to the PVN (Li et al., 2002 (link); Llewellyn et al., 2012 (link)). After sacrifice, the brain was removed and postfixed at 4°C for 4 h in 4% paraformaldehyde solution and placed in 20% sucrose. The brain was blocked in the coronal plane, and sections 30 μm in thickness were cut with a cryostat. The sections were incubated with 10% donkey serum in phosphate-buffered saline (PBS) for 1 h and were then incubated with a primary antibody against NPR-C (rabbit monoclonal antibody, 1:500, Abcam, Cambridge, MA) overnight at 4°C. After being washed with PBS, the sections were incubated with Cy3-conjugated donkey anti-rabbit secondary antibody (1:400, Jackson ImmunoResearch, West Grove, PA) for 2 h. The nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). The sections were coverslipped with fluoromounting-G (Southern Biotech, Birmingham, AL). Distribution of NPR-C by immunofluorescence and latexgreen within the PVN was viewed using an Olympus fluorescence microscope equipped with a digital camera (Qimaging, Canada).
Zheng H., Patel T.A., Liu X, & Patel K.P. (2023). C-type natriuretic peptide (CNP) in the paraventricular nucleus-mediated renal sympatho-inhibition. Frontiers in Physiology, 14, 1162699.
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10

Immunostaining of NKCC1 in Cells

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Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, blocked with 1% BSA, 10% normal donkey serum, 0.2% Triton X-100 in PBS. Incubation with the NKCC1 primary antibody (1:250; Chemicon, AB3560P) was performed for 16h at 4°C. Incubation with Cy3-conjugated donkey anti-rabbit secondary antibody (711-165-152, Jackson ImmunoResearch, USA) was performed for 60 min at room temperature. Samples were mounted with Vectashield antifade medium with DAPI (Vector laboratories, Burlingame, CA, USA). Confocal stacks were acquired with a Leica TSC SP-8 mounted on a Leica DMI6000 inverted microscope using an HC Plan Apochromat x63 objective.
Milićević N., Duursma A., Ten Asbroek A.L.M.A., Felder-Schmittbuhl M.P, & Bergen A.A. (2019). Does the circadian clock make RPE-mediated ion transport "tick" via SLC12A2 (NKCC1)?. Chronobiology international, 36(11).
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