|Immunofluorescence (Frozen)||1:100 - 1:400|
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.
Cover sections with 4% formaldehyde dilute in 1X PBS.
NOTE: Formaldehyde is toxic, use only in fume hood.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised July 2016
Protocol Id: 151
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro26 of mouse dopamine transporter/DAT protein.
Dopamine is a neurotransmitter that plays important roles in the brain, particularly in dopamine pathways that control the motivational component of reward-motivated behavior. These behavioral outputs are generated by the basal ganglia via its interaction with multiple brain areas that modulate sensorimotor, emotional, and cognitive information (1). The spatiotemporal dynamics of dopamine signaling are regulated by the dopamine transporter (DAT), a presynaptic transmembrane protein that drives the reuptake of dopamine released into the synaptic cleft. This process occurs in a sodium- and chloride-dependent manner, with DAT transporting two sodium cations down their concentration gradient, along with one chloride anion and the dopamine substrate. DAT is a member of the neurotransmitter sodium symporter (NSS) family, which also includes serotonin, norepinephrine, glycine, and γ-aminobutyric acid (GABA) transporters. The NSS family is part of the larger solute carrier 6 (SLC6) family of proteins, which in addition to neurotransmitters, also include transporters of amino acids, osmolytes, and energy metabolites (2-5). Mutations in the DAT gene, SLC6A3, have been linked to Parkinson’s disease (PD), infantile parkinsonism-dystonia (IPD), autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), major depressive disorder (MDD), and bipolar disorder (BP) (5-8). In addition to its role in the brain, DAT is expressed by lymphocytes and monocytes in the periphery, where it modulates immune function. Altered expression levels of DAT in the periphery is also a suggested biomarker for some cancers (9-12).
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