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Cy3 and cy5

Manufactured by Jackson ImmunoResearch
Sourced in Sweden

Cy3 and Cy5 are fluorescent dyes commonly used in molecular biology and immunoassay techniques. Cy3 emits light in the orange-red region of the visible spectrum, while Cy5 emits light in the far-red region. These dyes are often used for labeling proteins, nucleic acids, and other biomolecules to facilitate detection and visualization in various applications.

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3 protocols using cy3 and cy5

1

Immunohistochemistry Embryo Staining

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For immunohistochemistry embryos were fixed and stained according to standard procedures. Guinea pig anti-SIMU (30 (link)) and guinea pig anti-Drpr (32 (link)) were used at a 1:5000 and 1:100 concentrations, respectively. Rabbit anti-activated caspase 3 (Dcp-1) (Cell Signaling) and mouse anti-GFP (Roche) were used at 1:100 concentration. Rabbit anti-Crq antibody (1:500) is a gift from N. Franc. Rabbit anti-Srp antibody (1:100) is a gift from J. Casanova, K. Campbell and N. Martin. Rabbit anti-Peroxidasin antibody (1:2000) is a gift from Jiwon Shim. Fluorescent secondary antibodies (Cy3/and Cy5/Jackson ImmunoResearch; Alexa Fluor 488/Molecular Probes) were used at 1:200 dilutions. For TUNEL labeling embryos were re-fixed, washed and labeled with the In Situ Cell Death Detection kit (Roche) according to the manufacture instructions. Images were acquired on a confocal microscope Zeiss LSM 700 or on a Zeiss Axio Observer microscope equipped with an Apotome system using the AxioVision software. 75% Glycerol solution was used as the imaging medium.
Live imaging was carried out by dechorionating embryos (stage 15), mounting them under Halocarbon oil, injecting 2–3% egg volume of LysoTracker (Molecular Probes) as described in Ref. (33 (link)). Recording started 30 min following injection.
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2

Immunolabeling of Neuronal Structures

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Brain slices were fixed as above, followed by 30% sucrose for cryoprotection. Thick sections (35 μm) were obtained with a sliding microtome (Microm, HM 400). Using standard immunocytochemical techniques, free-floating sections were rinsed twice in PBS buffer followed by 50% alcohol. Non-specific binding was blocked as above and sections were exposed to Fluorescein Avidin D (Vector Laboratories) for visualizing biocytin-filled cells followed by primary antisera at 4°C for 12–48 hours with vesicular GABA transporter (VGAT) rabbit polyclonal, a gift from Dr. R Reimer, and gephyrin mouse monoclonal (Synaptic Systems). Some sections were exposed to secondary antibody-conjugates with indocarbocyanine and/or indodicarbocyanine (Cy3 and Cy5; 1:200 Jackson ImmunoResearch Lab), Alexa Fluor 568 and/or Alexa Fluor 488 (both Alexa fluorophores, 1:1000; Invitrogen Corporation) for 2 hours at room temperature to allow triple labeling when necessary. Table 1 provides a list of antibodies used, sources and concentrations. The specificity of BDNF and TrkB receptor antibodies used in this study has been supported in previous studies (Snapyan et al., 2009 (link); Bergami et al., 2013 (link)).
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3

Immunofluorescence Staining Protocol for α-Synuclein

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Immunofluorescence stainings were performed with sheep anti-TH (Abcam, cat# ab118 (working dilution 1:2000)), mouse anti α-syn (BD bioscience, cat# 610787 (working dilution 1:1000)) and rabbit anti-p129 α-syn (Abcam, Cat#51253 (working dilution 1:1000))
antibodies. The free-floating sections were washed thrice with PBS from anti-freeze solution.
The sections were then incubated in 0.025% Triton X-100 (Sigma Aldrich) in PBS containing 5% of normal serums (Vector Laboratories Inc, US) matching the species used to raise the secondary antibody for that proper stainings for 1 h. Primary antibodies were diluted in PBS and overnight incubated at 4 °C. The next day, the sections were washed thrice with PBS for 5 min, then incubated with Cy3 and Cy5 (working dilution 1:300) fluorophore conjugated secondary antibodies (Jackson Immunoresearch, Sweden). The sections were mounted on positive charged superfrost plus glass slides (ThermoScientific, US) and cover-slipped using PVA/DABCO. The immunofluorescence stainings were visualized on a Leica SP8 laserscanning confocal microscope (Leica, Germany).
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