In wild-type mice gold particles signaling -syntrophin decorate astrocytic endfoot membranes (arrowheads) peripheral to the endothelium (End) of cortical capillaries (A)

In wild-type mice gold particles signaling -syntrophin decorate astrocytic endfoot membranes (arrowheads) peripheral to the endothelium (End) of cortical capillaries (A). DAPC protein), but higher levels of AQP4. In agreement, depletion of dystrophin or -syntrophin C while causing a dramatic loss of AQP4 from endfoot membranes of brain astrocytes C had only modest or insignificant effect, respectively, on the AQP4 pool in endfoot membranes of Mller cells. Also, while polarization of brain macroglia was less affected by dystrophin depletion than by targeted deletion of -syntrophin, the reverse was true for retinal macroglia. These data indicate that the molecular scaffolding in perivascular endfeet is more complex than previously assumed and that macroglia are heterogeneous with respect to the mechanisms that dictate their polarization. (mice) (Adams et al., 2000) at 8C12 weeks of age were used in this study. The animals were allowed ad libitum access to food and drinking water. For immunofluorescence and quantitative immunogold analysis 4 animals of each genotype were analyzed. All experiments were approved by the institutions Animal Care and Use Committee. Antibodies 3-Methyladenine We used rabbit affinity-purified polyclonal antibodies against dystrophin (Dys331) (Kramarcy et al., 1994), -syntrophin (Syn259) (Peters et al., 1997), CD31 (BD Pharmingen, San Diego, CA), 3-Methyladenine and AQP4. Two different antibodies towards AQP4 were used: (1) antibody raised against AQP4 C-terminus (Millipore, Billerica, MA; for immunofluorescence), and (2) antibody raised against amino acid residues 249C323 (Sigma, St. Louis, MO; for immunogold cytochemistry). Immunocytochemistry Animals were deeply anesthetized by an i.p. injection of a mixture of chloral hydrate, magnesium sulfate, and pentobarbital (142, 70, and 32 mg/kg, respectively). Retinae and brain tissue were fixed by transcardiac perfusion (~10 ml/min) with 0.2% dextran (MW 70,000) in phosphate buffer (PB), followed by either phosphate-buffered 4% formaldehyde, pH 7.4, or bicarbonate-buffered 4% formaldehyde, pH 6.0, followed by 4% formaldehyde, pH 10.5 (pH shift protocol; 0.2% picric acid was added to both solutions) (Nagelhus et al., 1998). Light microscopic immunocytochemistry Light microscopic immunocytochemistry 3-Methyladenine was performed by using a method of indirect immunofluorescence. The concentrations of the antibodies were: Dys331, 6 g/ml, Syn259, 1.2 g/ml; anti-CD31, 2.5 g/ml; anti-AQP4, 2 g/ml. Antibodies were diluted in 0.01 M PB with 3% normal goat serum, 1% bovine serum albumin, 0.5% Triton X-100, and 0.05% sodium azide, pH 7.4. The primary antibodies were revealed by a carboxymethylindocyanine (Cy3) or (Cy5)-coupled donkey secondary antibody (1:1,000: Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Secondary antibodies were diluted in the same solution as the primary antibodies with the omission of sodium azide. Sections of retina and brain were viewed and photographed with a Zeiss LSM 5 Pascal confocal microscope (Carl Zeiss GmbH, Oberkochen, Germany). Sections from brain and retina of all genotypes were run in the same experiment to ensure comparable data. Furthermore all micrographs were as far as possible acquired with the same settings. Electron microscopic immunocytochemistry and morphological analysis For immunogold cytochemistry, small blocks of the eyecup and the parietal cortex were subjected to freeze substitution and infiltration in Lowicryl HM20 resin (Polysciences Inc., Warrington, PA, Cat 15924) (Schwarz and Humbel, 1989), before labeling with primary and secondary antibodies. Sections were incubated sequentially in the following solutions (at room temperature): (1) 50 mM glycine in Tris buffer (5 mM) containing 0.01% Triton X-100 and 50 mM NaCl (TBST; 10 min); (2) 0.2% milk powder in TBST (10 min); (3) primary antibody (anti-AQP4 from Sigma, 1.5 g/ml; anti–syntrophin, 12 g/ml) diluted in the solution used in the preceding step (overnight); (4) same solution as in step 2 2 (10 min 2); (5) gold-conjugated IgG (GAR15 nm for AQP4; 3-Methyladenine GAR10 nm Tpo for -syntrophin, Abcam, Cambridge, UK), diluted 1:20 in TBST containing milk powder and polyethylene glycol (0.5 mg/ml, 1 h). Finally, the sections were counterstained and examined in a.