Astrocyte purity was determined by immunofluorescence using antibodies specific for GFAP (Santa Cruz Biotechnology) and S100b (Sigma). Antibodies specific to CD11b (Serotec) and galactocerebroside (Sigma) were used to identify microglia or oligodendrocytes, respectively.
Ab levels in cell-culture supernatants and adherent cells were determined by immunoblotting with antibody 266 (against human Ab13–28) or 3D6 (against human Ab1–5; 2 g/ml; P. Seubert, Elan Pharmaceuticals).
Ab1–42 levels were measured with ELISA using capture antibody 21F12 (against human Ab33–42) and biotinylated reporter antibody 3D6.
Sections of murine brain expressing human APP were incubated with or without adult astrocytes then fixed, immunostained with 3D6
antibody (1:1000) and developed with DAB. For confocal microscopy, sections similarly incubated with astrocytes were fixed and immunolabeled with rabbit antibody against human Ab1282 (1:1000; D. Selkoe, Harvard University, Boston, Massachusetts) and goat antibody against S100 (1:1000).