Large scale brain mosaic showing the distribution of the dopamine receptor-associated phosphoprotein DARRP-32 (green) and the vesicular monamine transporter VMAT-2 (red) in a hemisection through the anterior neostriatum of a dopamine transporter knock out mouse. This image was generated by tiling 45 by 65 (X, Y axes) frames of maximum projection images in the Z-axis. This image has been downsampled from the raw data image which can be accessed using the link provided to the Cell Centered Database.
Transgenic mice (strain DATKO) from Duke University were perfused with aldehyde fixatives (4% paraformaldehyde + 0.1% gluteraldehyde), sectioned on Vibratome (thickness, 80 µm), and rinsed in phosphate buffered saline (PBS,3 x 10min) and incubated in blocking buffer (PBS, 3% normal donkey serum, 1% fish skin gelatin, 0.3% Triton X-100, 1% bovine serum albumin). Tissue sections incubated on shaker overnight, 4° C in primary antibodies diluted in blocking buffer as follows: anti-VMAT-2; Host = guinea pig; 1:500 (Oncogene, catalog # 503-01-50); anti-DARPP-32; Host = mouse; 1:500 (BD Transduction Laboratories, catalog #611520). Following 3 x 10' rinses, tissue was incubated for 2hr, RT, on a shaker in secondary antibodies diluted at 1:100 in working buffer (donkey-anti-mouse AF488 [Molecular Probes, Cat #A21202] and donkey-anti-guinea pig RRX [Jackson Immunoresearch Laboratories, Inc]. Following rinses in PBS, sections were mounted on slides with gelvatol. Individual images were gathered using a BioRad RTS 2000 Multiphoton with a Nikon PlanApo objective (N.A. 1.45).
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