Immunogold cytochemistry in neuroscience

Amiry-Moghaddam, Mahmood; Ottersen, Ole Petter

doi:10.1038/nn.3418

Cited by 49 publications

(64 citation statements)

References 67 publications

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“…For the interpretation of the labeling in FRIL, it should be kept in mind that the immunogold particle can be located anywhere within a hemisphere with a radius of 20-25 nm from the antigen due to the flexible complex formed by the primary and secondary antibody 26 . For further information on the theory and practice of FRIL and related techniques, we refer the reader also to other methodological articles 27,28 .…”

Section: Discussionmentioning

confidence: 99%

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Schönherr

Seewald

Kasugai

et al. 2016

JoVE

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Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre-and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

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Section: Discussionmentioning

confidence: 99%

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Schönherr

Seewald

Kasugai

et al. 2016

JoVE

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“…25 As a control, the secondary gold-labeled antibody was incubated with platelets alone but no gold nanoparticles were detected in the vicinity of the plasma membrane (data not shown).…”

Section: Zooming Into Intact Platelets With Cryo-electron Tomographymentioning

confidence: 96%

Toward correlating structure and mechanics of platelets

Sorrentino

Studt

Horev

et al. 2016

Cell Adhesion & Migration

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The primary physiological function of blood platelets is to seal vascular lesions after injury and form hemostatic thrombi in order to prevent blood loss. This task relies on the formation of strong cellular-extracellular matrix interactions in the subendothelial lesions. The cytoskeleton of a platelet is key to all of its functions: its ability to spread, adhere and contract. Despite the medical significance of platelets, there is still no high-resolution structural information of their cytoskeleton. Here, we discuss and present 3-dimensional (3D) structural analysis of intact platelets by using cryoelectron tomography (cryo-ET) and atomic force microscopy (AFM). Cryo-ET provides in situ structural analysis and AFM gives stiffness maps of the platelets. In the future, combining highresolution structural and mechanical techniques will bring new understanding of how structural changes modulate platelet stiffness during activation and adhesion.

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“…A common feature of all these techniques is that they shift the n from the animal or tissue level to the cellular or even subcellular level, and invariably yield data with a nested structure. For instance, super resolution light microscopy allows imaging and advanced understanding of neuronal compartments 18 , immunogold cytochemistry allows determination of subcellular localization of pro teins 19 , and recent advances in optogenetics and optopharmacology facilitate selective control of electrical and protein activity, respec tively, in circuits, individual cells or subcellular compartments 20,21 . All these techniques concern the collection of multiple observations from one cell, thereby yielding nested data.…”

Section: Box 3 Estimating the Power To Detect An Experimental Effectmentioning

confidence: 99%