Subcellular localization of GABA receptors in the central nervous system using post-embedding immunohistochemistry.
The following detailed protocol can be applied to demonstrate the localization of GABA receptors in CNS neurons at the ultrastructural level. While others have investigated receptors at the electron microscopic level using immunocytochemical techniques, the appearance of the tissue is usually poor and analyses of the distribution of receptors is limited. The methodology described in this paper allows for optimal preservation of the tissue while retaining immunogenicity. It does this, in part, by utilizing a balanced salt solution washout in conjunction with fixation. When the ionic composition of a fixative solution differs from extracellular fluids, like in most fixation protocols for electron microscopy, ultrastructural changes may occur in the tissue. Balanced salt solutions, like the Tyrode solution used here, helps maintain the normal extracellular environment allowing the fixing agent to reach sufficient concentration to bring about permanent and more optimal fixation even when reduced amounts of glutaraldehyde are required to preserve antigenicity. Therefore, unlike many protocols for post-embedding immunoelectronmicroscopy, this method allows for superior preservation of tissue ultrastructure compared to results previously published by others.
Department of Biology and Cell/Molecular Physiology, University of North Carolina--Chapel Hill, Chapel Hill, NC 27599, USA.
Wendy L
WL
Heck
Arkadiusz
A
Slusarczyk
Ana Maria
AM
Basaraba
Laura
L
Schweitzer
D000818:Animals; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D003712:Dendrites / Q000378:metabolism / Q000648:ultrastructure; D007150:Immunohistochemistry / Q000379:methods*; D007552:Isotonic Solutions; D008854:Microscopy, Electron / Q000379:methods*; D010477:Perfusion / Q000379:methods*; D016613:Plastic Embedding / Q000379:methods*; D017729:Presynaptic Terminals / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D018079:Receptors, GABA / Q000378:metabolism*; D011963:Receptors, GABA-A / Q000378:metabolism; D018080:Receptors, GABA-B / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism* / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology; D016707:Tissue Fixation / Q000379:methods*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D007552:Isotonic Solutions; D018079:Receptors, GABA; D011963:Receptors, GABA-A; D018080:Receptors, GABA-B; C037692:Tyrode's solution; D005680:gamma-Aminobutyric Acid