Confocal immunofluorescent analysis of Doublecortin (A8L1U) Rabbit mAb (green) and GFAP (GA5) Mouse mAb (Alexa Fluor® 555 Conjugate) #3656 (red) staining in the subventricular zone of adult mouse brain. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Confocal immunofluorescent analysis of P5 mouse cerebellum using Doublecortin (A8L1U) Rabbit mAb (green) and GFAP (GA5) Mouse mAb #3670 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
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
Doublecortin (A8L1U) Rabbit mAb recognizes endogenous levels of total doublecortin protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human doublecortin protein.
Mutations in Doublecortin cause Lissencephaly (smooth brain), a neuronal migration disorder characterized by epilepsy and mental retardation (1). Doublecortin is a microtubule associated protein that stabilizes and bundles microtubules. A conserved doublecortin domain mediates the interaction with microtubules, and interestingly most missense mutations cluster in this domain (2). Kinases JNK, CDK5 and PKA phosphorylate doublecortin. JNK phosphorylates Thr321, Thr331 and Ser334 while PKA phosphorylates Ser47 and CDK5 phosphorylates Ser297 (3-5). Phosphorylation of Ser297 lowers the affinity of doublecortin to microtubules. Furthermore, mutations of Ser297 result in migration defects (5).
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