A semi-correlative technique for the subcellular localization of proteins in Drosophila synapses. / Jiao, Wei; Shupliakov, Andrey; Shupliakov, Oleg.
In: Journal of Neuroscience Methods, Vol. 185, No. 2, 15.01.2010, p. 273-279.Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - A semi-correlative technique for the subcellular localization of proteins in Drosophila synapses
AU - Jiao, Wei
AU - Shupliakov, Andrey
AU - Shupliakov, Oleg
PY - 2010/1/15
Y1 - 2010/1/15
N2 - The neuromuscular junction (NMJ) in Drosophila larvae serves as an important model system to study synaptic vesicle recycling, plasticity, development, and organelle trafficking in nerve terminals. Here we provide a protocol that reliably localizes proteins subcellularly in this system in combination with good preservation of the ultrastructure of the NMJ. In this protocol, the NMJs are cut open to increase access of reagents to the interior of the synapse, stained with primary antibodies followed by secondary antibodies conjugated to Fluoronanogold particles to allow selection of the best-stained areas for electron microscopic (EM) analysis using confocal microscopy. To improve visualization at the EM level, the nanogold particles are silver enhanced. Good penetration of the antibodies in this protocol allows for a three-dimensional reconstruction of the labeling pattern from serial ultrathin sections. To explore the utility and resolution of this approach, we determined the ultrasturcutral localization of the cell adhesion molecule Fascicline II (Fas II) at rest using an antibody raised against the cytoplasmic epitope. We observed that Fas II-ir is accumulated at the cytoplasmic surfaces of pre- and postsynaptic elements of the synapse at equal distances from the membranes, thus supporting the model that it has a similar orientation in these opposing synaptic structures. Fas II-ir delineates active zones and is compartmentalized from CSP, Dap160, and Bruchpilot immunolabeling. We conclude that Fas II immunolabeling in Drosophila can be reliably used to evaluate changes in the size of the active zone region at rest in genetic and functional experiments.
AB - The neuromuscular junction (NMJ) in Drosophila larvae serves as an important model system to study synaptic vesicle recycling, plasticity, development, and organelle trafficking in nerve terminals. Here we provide a protocol that reliably localizes proteins subcellularly in this system in combination with good preservation of the ultrastructure of the NMJ. In this protocol, the NMJs are cut open to increase access of reagents to the interior of the synapse, stained with primary antibodies followed by secondary antibodies conjugated to Fluoronanogold particles to allow selection of the best-stained areas for electron microscopic (EM) analysis using confocal microscopy. To improve visualization at the EM level, the nanogold particles are silver enhanced. Good penetration of the antibodies in this protocol allows for a three-dimensional reconstruction of the labeling pattern from serial ultrathin sections. To explore the utility and resolution of this approach, we determined the ultrasturcutral localization of the cell adhesion molecule Fascicline II (Fas II) at rest using an antibody raised against the cytoplasmic epitope. We observed that Fas II-ir is accumulated at the cytoplasmic surfaces of pre- and postsynaptic elements of the synapse at equal distances from the membranes, thus supporting the model that it has a similar orientation in these opposing synaptic structures. Fas II-ir delineates active zones and is compartmentalized from CSP, Dap160, and Bruchpilot immunolabeling. We conclude that Fas II immunolabeling in Drosophila can be reliably used to evaluate changes in the size of the active zone region at rest in genetic and functional experiments.
KW - Drosophila larvae
KW - Electron microscopy
KW - Immunocytochemistry
KW - Neuromuscular junction
KW - Synapse
UR - http://www.scopus.com/inward/record.url?scp=72249090840&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2009.10.010
DO - 10.1016/j.jneumeth.2009.10.010
M3 - Article
C2 - 19850080
AN - SCOPUS:72249090840
VL - 185
SP - 273
EP - 279
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
SN - 0165-0270
IS - 2
ER -
ID: 40830624