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Heat-mediated antigen retrieval is recommended prior to staining, using a 10mM citrate buffer, pH 6.0, for 10 minutes followed by cooling at room temperature for 20 min. Following antigen retrieval, incubate samples with primary antibody for 10 min at room temperature. A suggested positive control is pancreas tissue.
GAD-65 and GAD-67 are members of the group II decarboxylase family of proteins and are responsible for catalyzing the rate limiting step in the production of GABA (gamma-aminobutyric acid) from L-glutamic acid. Although both GADs are found in the brain, GAD-65 localizes to synaptic vesicle membranes in nerve terminals, while GAD-67 is distributed throughout the cell. GAD-67 is responsible for the basal levels of GABA synthesis. In the case of a heightened demand for GABA in neurotransmission, GAD-65 will transiently activate to assist in GABA production. The loss of GAD-65 is detrimental and can impair GABA neurotransmission, however the loss of GAD-67 is lethal. Due to alternative splicing, two isoforms exist for GAD-67: the predominant GAD-67 form and the minor GAD-25 form.
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