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Description
Rat hippocampal neurons grown in vitro, fixed after 23 days in culture, and immunostained for MAP2 (red) to reveal the dendritic arbor, and localization of α Calcium Calmodulin Kinase II (green), a multifunctional kinase that has high concentrations at excitatory postsynaptic sites and presynaptic terminals, and is involved in regulating synaptic plasticity. Detailed methods: Embryonic rat hippocampal neurons were prepared as previously described (see Kaech and Banker, 2006, Nat Protoc). Cells were prepared for fluorescent staining as previously described (Withers and Banker, 1998, in Culturing Nerve Cells, MIT Press). Briefly, cells were fixed (4% formaldehyde, 4% sucrose in phosphate buffered saline, pH 7.4, warmed to 37°C prior to fixation, 15 minutes), permeabilized with 0.25% Triton (7 minutes) and immunostained for alpha-CamKII (clone 6G9, Abcam, with Alexa 488 conjugated secondary, excitation, 494, emission, 519 [Invitrogen, Molecular Probes]) and MAP2 (Ab266 from S. Halpain, with DyLight549 conjugated secondary, excitation, 555, emission, 568, [Jackson Immunoresearch]). Images were acquired with a Leica DMRA microscope with a mercury arc lamp, a 40X lens (HCX PL Fluotar, NA 0.75), Leica GFP filter set (excitation, BP 470/40; dichromatic mirror, 500; suppression filter, BP 525/50); Leica N3 filter set (excitation, BP546/12; dichromatic mirror, 565, suppression filter, BP 600/40), Photometrics CoolSnap ES CCD camera and MetaMorph software. This merged image was generated with the MetaMorph color combine function. Research Data Curation Program, UC San Diego, La Jolla, 92093-0175 (https://lib.ucsd.edu/rdcp) Brandner, Dieter; Withers, Ginger (2021). CIL:8728, Rattus, multipolar neuron. In Cell Image Library. UC San Diego Library Digital Collections. Dataset. https://doi.org/10.6075/J0PV6KCH
Type
image
Identifier
ark:/20775/bb32498913
Language
No linguistic content
Subject
Dendrite development Calmodulin-dependent protein kinase activity Cellular developmental process Establishment or maintenance of cell polarity Axon Microtubule cytoskeleton Multipolar neuron Synapse Dendrite Rattus Cell Image Library Group ID: 7783
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