donkey anti goat cy3, by Jackson ImmunoResearch - Product details

1

Visualizing Retinal Cell Types in Mice

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Sert^Cre/+ Tau^{mGFP-NLS-LacZ} P7 mice were euthanized with an overdose of pentobarbital-xylazine and perfused transcardially with 4% PFA in 0.12 M PB. Eyes were postfixed overnight in the same fixative, cryoprotected with 10% sucrose in PB during 2 days. Eyes were then embedded with 7.5% gelatin and 10% sucrose in PB, frozen 1 minute in isopentane at −55 °C, sectioned with a cryostat (20 µm; Leica CM 3000) and collected on superfrost slides. Slides were stored at −80 °C.
Sections were blocked with PGTx (0.2% gelatin and 0.25% Triton X-100 in PBS). Antibodies were diluted in PGTx: Rabbit anti-βGal (1/5000, Rockland), Goat anti-ChAT (1/200, Chemicon), Donkey anti-rabbit 488 and Donkey anti-goat Cy3 (1/500; Jackson Immunoresearch).
For retinal whole-mount immunostaining, retinas were first permeabilized in 1% Triton X-100 for 30 minutes, blocked in PHTx (0.1% Triton X-100 10% horse serum in PBS). Antibodies were diluted in PHTx: Rabbit anti-βGal (1/5000, Rockland), Goat anti-Brn3 (1/200, C-13, Santa Cruz), Rabbit anti-RBPMS (1/500, Phosphosolutions, # 1830), Donkey anti-rabbit AlexaFluor488, Donkey anti-chicken AlexaFluor488, Donkey anti-rabbit Cy3 and Donkey anti-goat Cy3 (1/200, Jackson). The anti-Brn3 antibody marks the Brn3a, b and c, leading to the labeling of 80% of RGCs^{31 (link)}.

Assali A., Le Magueresse C., Bennis M., Nicol X., Gaspar P, & Rebsam A. (2017). RIM1/2 in retinal ganglion cells are required for the refinement of ipsilateral axons and eye-specific segregation. Scientific Reports, 7, 3236.

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2

Intraoral Stimulation-Induced c-Fos Mapping

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Individual mice were implanted with an intraoral cannula^{28 (link)} 3 days before c-Fos induction. On the day of experiments, mice were anaesthetized with urethane (1.6 mg/g) and the trachea was cannulated to aid breathing during oral stimulus presentation. Tastants were perfused into the mouth through the intraoral cannula for 1.5 hours at a rate of ~6 ml/hr. Mice were allowed to rest for 30 minutes and processed for immunostaining as previously described. The brains were sectioned coronally at 100 μm, and labeled with goat anti-c-Fos (Santa Cruz, sc-52-G) overnight; Alexa 488 donkey anti-goat or cy3 donkey anti-goat (Jackson immunoResearch) were used to visualize c-Fos expression. All images were taken using an Olympus FluoView 1000 confocal microscope.

Peng Y., Gillis-Smith S., Jin H., Tränkner D., Ryba N.J, & Zuker C.S. (2015). Sweet and bitter taste in the brain of awake behaving animals. Nature, 527(7579), 512-515.

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3

Intraoral Stimulation-Induced c-Fos Mapping

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Individual mice were implanted with an intraoral cannula^{28 (link)} 3 days before c-Fos induction. On the day of experiments, mice were anaesthetized with urethane (1.6 mg/g) and the trachea was cannulated to aid breathing during oral stimulus presentation. Tastants were perfused into the mouth through the intraoral cannula for 1.5 hours at a rate of ~6 ml/hr. Mice were allowed to rest for 30 minutes and processed for immunostaining as previously described. The brains were sectioned coronally at 100 μm, and labeled with goat anti-c-Fos (Santa Cruz, sc-52-G) overnight; Alexa 488 donkey anti-goat or cy3 donkey anti-goat (Jackson immunoResearch) were used to visualize c-Fos expression. All images were taken using an Olympus FluoView 1000 confocal microscope.

Peng Y., Gillis-Smith S., Jin H., Tränkner D., Ryba N.J, & Zuker C.S. (2015). Sweet and bitter taste in the brain of awake behaving animals. Nature, 527(7579), 512-515.

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4

Immunostaining of Cytoskeletal Proteins

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Cells were fixed in 4% paraformaldehyde
in PBS for 15 min, permeabilized with 0.1% Triton X for 10 min, and
blocked with 1% BSA in PBS for 30 min. The cells were then incubated
with primary antibody for 1 h at room temperature and overnight at
4 °C, followed by a 2 h incubation at room temperature with secondary
antibody and a 10 s incubation with DAPI for nuclear staining. Primary
antibodies were monoclonal mouse anti-α-smooth muscle actin
(α-SMA, Sigma, 1:500), polyclonal rabbit antidesmin (Abcam,
1:80), and antihuman lumican (R&D Systems, 1:400). Secondary antibodies
were cy2 donkey antimouse (1:200), cy3 donkey antirabbit (1:200),
and cy3 donkey antigoat (1:200), all from Jackson Immunoresearch Laboratories.

Saums M.K., Wang W., Han B., Madhavan L., Han L., Lee D, & Wells R.G. (2014). Mechanically and Chemically Tunable Cell Culture System for Studying the Myofibroblast Phenotype. Langmuir, 30(19), 5481-5487.

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5

Aβ-Mediated Interaction Between P. gingivalis and Amyloid

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Recombinant Aβ (1-42) Ultra-Pure, Ammonium Hydroxide (rProtein) was prepared as stock solutions (1 mg/ml) in 1% NaNH₄. P. gingivalis was washed in PBS and incubated at 10⁸ CFU/ml in Aβ (10, 30, and 100 μg/ml) for 1 hour at RT and ambient oxygen. For IHC, a 10-μl solution was dried on a SuperFrost Plus glass slide (VWR) fixed in 4% paraformaldehyde for 10 min. Slide was then rinsed with PBS and dH₂O, exposed to formic acid, and, after washing with PBS, incubated for 2 hours in PBS, 0.3% Triton X-100, and primary antibodies CAB102 (1:1000) and 4G8 (1:1000; BioLegend). Fluorescence labeling was performed with Alexa Fluor 488 donkey anti-rabbit (1:200; Jackson ImmunoResearch) and Cy3 donkey anti-goat (1:200; Jackson ImmunoResearch) in PBS. Slides were mounted with ProLong Gold Antifade (Thermo Fisher Scientific). Images were taken on an Olympus BX61 microscope with a Zyla 5.5 sCMOS camera (Andor).

Dominy S.S., Lynch C., Ermini F., Benedyk M., Marczyk A., Konradi A., Nguyen M., Haditsch U., Raha D., Griffin C., Holsinger L.J., Arastu-Kapur S., Kaba S., Lee A., Ryder M.I., Potempa B., Mydel P., Hellvard A., Adamowicz K., Hasturk H., Walker G.D., Reynolds E.C., Faull R.L., Curtis M.A., Dragunow M, & Potempa J. (2019). Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors. Science Advances, 5(1), eaau3333.

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6

Immunohistochemical Analysis of Embryonic Tissues

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Embryos were fixed in 4% paraformaldehyde (Sigma) in PBS for 2 h at 4°C, cryoprotected in 30% sucrose in PBS overnight and frozen in NEG-50 Frozen Section Medium (Thermo Fisher). Frozen embryos were thin-sectioned to yield 20 μM transverse sections with a cryostat. Antibody staining was performed on cryostat sections after blocking in 5% NGS in PBS containing 0.1% Triton X-100 or with 2% horse serum in PBS containing 0.1% Triton X-100 (for all anti-goat antibodies) for 1h at room temperature. Sections were then incubated with primary antibodies overnight at 4°C. After three washes in PBS, sections were incubated with species-specific secondary antibodies conjugated to fluorophores at room temperature for 2 hr. Antibodies used: rabbit anti-Ndfip1 (1:100, Sigma #HPA009682), mouse anti-TAG1 (1:100, DSHB #4D7), goat anti-Robo3 (1:200, R&D systems #AF3076), goat anti-Robo1 (1:200, R&D systems #AF1749), rabbit anti-GFP (1:1000, Invitrogen #A11122), rat anti-L1CAM (1:300, Millipore #MAB5272), Alexa488 goat anti-rabbit (Invitrogen, 1:500 #A11034), Cy3 goat anti-mouse (1:1000, Jackson Immunoresearch # 115-165-003), Cy3 donkey anti-goat (1:400, Jackson Immunoresearch #705-165-003), and Alexa633 goat anti-Rat (1:500, Invitrogen #A-21094).

Gorla M., Santiago C., Chaudhari K., Layman A.A., Oliver P.M, & Bashaw G.J. (2019). Ndfip Proteins Target Robo Receptors for Degradation and Allow Commissural Axons to Cross the Midline in the Developing Spinal Cord. Cell reports, 26(12), 3298-3312.e4.

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7

Multimodal Analysis of Tissue Markers

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Previously described IHC procedures [45 (link)] were used on 4 µm tissue sections. Specifically, after citrate antigen retrieval, sections were exposed to 0.3% H₂O₂ in methanol, TNT blocking buffer (Perkin Elmer), primary antibodies (anti-CD31 Ab (ab28364, Abcam), anti-CLEC4F Ab (AF2784, R&D Systems)) and detection agents (CD31: polymer HRP anti-rabbit followed by opal 520 fluorophore amplification reagent (PerkinElmer); CLEC4F: Cy-3 donkey anti-goat (Jackson Immuno Research). Nuclear stain: DAPI. Previously described RNAScope method [1 (link)] was used on 4 µm tissue sections using probes from ACD Bio.

Zhao Q., Molina-Portela M.D., Parveen A., Adler A., Adler C., E H., Wang W., Ni M., Wei Y., Atwal G., Mohrs M., Thurston G, & Eichten A. (2020). Heterogeneity and chimerism of endothelial cells revealed by single-cell transcriptome in orthotopic liver tumors. Angiogenesis, 23(4), 581-597.

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8

Immunofluorescence Analysis of Cellular Markers

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Cells on a 24-well plate were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS containing 0.1% Triton X-100 (PBS-T) two times for 3 min each and then blocked/permeabilized with PBS-T containing 1% BSA for 1 h. Then, the wells were prepared for immunofluorescence analysis as previously described (20 (link)). Fluorescence micrographs were obtained with the microscope Zeiss Imager M1 (Carl Zeiss Microscopy GmbH, Germany) equipped with FITC, rhodamine, and FURA filters. Primary antibodies used were: MuRF2 (18 (link)), MyoD (sc-31940), Ki-67 (NB110-89717), and TCF-4 (sc-13027). Secondary antibodies used were: Cy3 Donkey Anti-rabbit (Jackson ImmunoResearch #711-165-152), Cy3 Donkey Anti-goat (Jackson ImmunoResearch #705-175-147), and Cy2 Goat Anti-rabbit (Jackson ImmunoResearch #111-225-008). Digital images from a Zeiss Axiocam MRn were exported as TIFF files with Axiovision 4.6 software without any subsequent processing in parameters such as contrast and brightness.

Silvestre J.G., Baptista I.L., Silva W.J., Cruz A., Silva M.T., Miyabara E.H., Labeit S, & Moriscot A.S. (2019). The E3 ligase MuRF2 plays a key role in the functional capacity of skeletal muscle fibroblasts. Brazilian Journal of Medical and Biological Research, 52(9), e8551.

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9

Immunohistochemical Analysis of Embryonic Tissues

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Embryos were fixed in 4% paraformaldehyde (Sigma) in PBS for 2 h at 4°C, cryoprotected in 30% sucrose in PBS overnight and frozen in NEG-50 Frozen Section Medium (Thermo Fisher). Frozen embryos were thin-sectioned to yield 20 μM transverse sections with a cryostat. Antibody staining was performed on cryostat sections after blocking in 5% NGS in PBS containing 0.1% Triton X-100 or with 2% horse serum in PBS containing 0.1% Triton X-100 (for all anti-goat antibodies) for 1h at room temperature. Sections were then incubated with primary antibodies overnight at 4°C. After three washes in PBS, sections were incubated with species-specific secondary antibodies conjugated to fluorophores at room temperature for 2 hr. Antibodies used: rabbit anti-Ndfip1 (1:100, Sigma #HPA009682), mouse anti-TAG1 (1:100, DSHB #4D7), goat anti-Robo3 (1:200, R&D systems #AF3076), goat anti-Robo1 (1:200, R&D systems #AF1749), rabbit anti-GFP (1:1000, Invitrogen #A11122), rat anti-L1CAM (1:300, Millipore #MAB5272), Alexa488 goat anti-rabbit (Invitrogen, 1:500 #A11034), Cy3 goat anti-mouse (1:1000, Jackson Immunoresearch # 115-165-003), Cy3 donkey anti-goat (1:400, Jackson Immunoresearch #705-165-003), and Alexa633 goat anti-Rat (1:500, Invitrogen #A-21094).

Gorla M., Santiago C., Chaudhari K., Layman A.A., Oliver P.M, & Bashaw G.J. (2019). Ndfip Proteins Target Robo Receptors for Degradation and Allow Commissural Axons to Cross the Midline in the Developing Spinal Cord. Cell reports, 26(12), 3298-3312.e4.

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10

Visualizing Stress-Induced Neural Activity

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To detect general anesthesia-activated neurons, animals were anesthetized with isoflurane, or ketamine/xylazine, or dexmedetomidine, for two hours then transcardially perfused with 10% sucrose in cold phosphate buffer saline (PBS, pH 7.4) followed by 4% cold paraformaldehyde (PFA) fixation solution. To detect restraint stress-activated neurons, mice were placed in the restrainer for 90 min (TV-150, Braintree scientific Inc). All brains were post-fixed in PFA overnight at 4°C, cryoprotected in 30% sucrose PBS solution for 2–3 days at 4°C, frozen in O.C.T compound (Tissue-Tek, Sakura), and then stored at −80°C until sectioning. Floating brain sections (80 µm) were stained using standard immunofluorescent protocol. The primary antibody used were: goat anti-Fos (Santa Cruz Biotechnology, sc520g, 1:300)⁵⁰, rabbit anti-Fos (Cell Signaling, #2250, 1:1500)⁵¹, anti-Neurotensin (ImmunoStar, 20072)⁵². The secondary antibody used are: Alexa Fluor 488 donkey anti-goat (Jackson ImmunoResearch, 705-545-147, 1:500)⁵³, Alexa Fluor 647 anti-rabbit (Jackson ImmunoResearch, 711-605-152, 1:500)⁵⁴, and Cy3 donkey anti-goat (Jackson ImmunoResearch, 705-165-147, 1:500)⁵⁵ (see Life Sciences Reporting Summary).

Hua T., Chen B., Lu D., Sakurai K., Zhao S., Han B.X., Kim J., Yin L., Chen Y., Lu J, & Wang F. (2020). General Anesthetics Activate a Potent Central Pain-Suppression Circuit in the Amygdala. Nature neuroscience, 23(7), 854-868.

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Donkey anti goat cy3

Lab products found in correlation

40 protocols using donkey anti goat cy3

Visualizing Retinal Cell Types in Mice

Intraoral Stimulation-Induced c-Fos Mapping

Intraoral Stimulation-Induced c-Fos Mapping

Immunostaining of Cytoskeletal Proteins

Aβ-Mediated Interaction Between P. gingivalis and Amyloid

Immunohistochemical Analysis of Embryonic Tissues

Multimodal Analysis of Tissue Markers

Immunofluorescence Analysis of Cellular Markers

Immunohistochemical Analysis of Embryonic Tissues

Visualizing Stress-Induced Neural Activity

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