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Appropriate fluorescent secondary antibodies

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Appropriate fluorescent secondary antibodies are laboratory reagents designed to detect and visualize target proteins in various biological applications. These antibodies are conjugated with fluorescent dyes that emit light at specific wavelengths, enabling the identification and localization of the target proteins within a sample. The core function of these reagents is to provide a sensitive and specific detection method for researchers and scientists working in the fields of immunology, cell biology, and molecular biology.

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Lab products found in correlation

Lsm 700 confocal microscope, by Zeiss (2 mentions) Dapi vectorshield mounting medium, by Vector Laboratories (1 mentions) Goat anti epha2, by R&D Systems (1 mentions) Gapdh mouse monoclonal antibody, by Abcam (1 mentions) β3 tubulin rabbit polyclonal and mouse monoclonal antibody, by Fortrea (1 mentions) Fluoro jade c staining kit, by Biosensis (1 mentions) Bz 9000, by Keyence (1 mentions) Neuronal nuclei (neun), by Abcam (1 mentions)

4 protocols using appropriate fluorescent secondary antibodies

1

Immunostaining of Hypothalamic Slices

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After 6 days ex vivo, hypothalamic slices were washed with PBS and fixed by immersion in 4% PFA for 2 hours at room temperature. Slices were labeled using antibodies against vimentin (V9, Sigma Aldrich) and Darpp‐32 (19A3, Cell Signaling) and appropriate fluorescent secondary antibodies (Jackson ImmunoResearch). The membranes carrying the slices were transferred onto microscope slides and mounted using mounting medium containing Hoechst. Labelling was visualized by fluorescence microscopy.
Stoney P.N., Helfer G., Rodrigues D., Morgan P.J, & McCaffery P. (2015). Thyroid hormone activation of retinoic acid synthesis in hypothalamic tanycytes. Glia, 64(3), 425-439.
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2

Immunolocalization of EphA2 and ephrin-A5 in mouse lens

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Lens capsule flat mounts with attached epithelial and peripheral fiber cells were prepared from postnatal day 21 (P21) non-GFP WT mouse lenses as previously described (Cheng et al., 2013 (link); Cheng and Gong, 2011 (link)). Briefly, freshly dissected lenses were flash fixed in cold 100% methanol for 45 seconds. Lens capsules (along with attached epithelial and peripheral fiber cells) was gently dissected away from the fiber cell mass using radial cuts. Capsules were blocked (10% normal donkey serum and 0.3% Triton X-100) for 1 hour at room temperature before staining with goat anti-EphA2 (R&D Systems) and rabbit anti-ephrin-A5 (Invitrogen) primary antibody overnight at 4°C. Primary antibody incubation was followed washing and then incubation in appropriate fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories) for 2 hours at room temperature. Lens capsules were then washed and flat mounted with DAPI VectorShield mounting medium (Vector Laboratories, Inc.). Z-stack images were collected by a Zeiss LSM700 confocal microscope, and 3D images were reconstructed from z-stack data collected at 0.38 μm steps using the ZEN software. Antibody specificity was tested previously using knockout tissues (Cheng and Gong, 2011 (link)).
Cheng C., Fowler V.M, & Gong X. (2017). EphA2 and ephrin-A5 are not a receptor-ligand pair in the ocular lens. Experimental eye research, 162, 9-17.
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3

Antibody Characterization for Neuronal Cytoskeleton

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Rabbit polyclonal antibody against MAP2 was provided by Itzhak Fischer (Drexel University College of Medicine, Philadelphia, PA; Nunez and Fischer, 1997 (link)). Other antibodies were purchased as noted: detyrosinated tubulin rabbit polyclonal antibody (Millipore), acetylated tubulin mouse monoclonal antibody (Sigma-Aldrich), tyrosinated tubulin YL1/2 rat monoclonal antibody (Accurate Scientific, Westbury, NY), β3-tubulin rabbit polyclonal and mouse monoclonal antibody (Covance, Princeton, NJ), β-tubulin fluorescein isothiocyanate–conjugated monoclonal mouse antibody (Sigma-Aldrich), kinesin-5 phosphorylated at Thr-926 rabbit polyclonal antibody (Phospho Solutions, Aurora, CO), total kinesin-5 rabbit polyclonal antibody (Abcam, Cambridge, UK), and mouse monoclonal GAPDH antibody (Abcam). Appropriate fluorescent secondary antibodies were purchased from Jackson Immunoresearch (West Grove, PA).
Kahn O.I., Sharma V., González-Billault C, & Baas P.W. (2015). Effects of kinesin-5 inhibition on dendritic architecture and microtubule organization. Molecular Biology of the Cell, 26(1), 66-77.
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4

Immunohistochemical Analysis of Neuronal Markers

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Immunohistochemistry was conducted as routinely executed (Chen, Zhang, et al., 2015 (link); Huang et al., 2015 (link)). The rat pups were perfused under anesthesia with PBS followed by 4% formaldehyde. The brains were removed for postfixation in formalin. The paraffin-embedded brains were then sectioned into 10 µm slices via cryostat. The brain slices were then evaluated with immunohistochemistry and a Fluoro-Jade C staining kit (Biosensis). The specific cellular marker antibody used was NeuN (1:100; Abcam). The brain slices were then incubated with the appropriate fluorescent secondary antibodies (Jackson ImmunoResearch). The brain slices were then visualized with a fluorescent microscope (Olympus BX51 and Keyence BZ-9000) under 20×, 40×, and 60 × magnifications with an aperture of 0.75, 0.95, and 0.95, respectively.
Dixon B.J., Chen D., Zhang Y., Flores J., Malaguit J., Nowrangi D., Zhang J.H, & Tang J. (2016). Intranasal Administration of Interferon Beta Attenuates Neuronal Apoptosis via the JAK1/STAT3/BCL-2 Pathway in a Rat Model of Neonatal Hypoxic-Ischemic Encephalopathy. ASN NEURO, 8(5), 1759091416670492.
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