Mouse anti neun or goat anti map2 antibodies

As previously described (Buscemi et al., 2007 (link); Hui et al., 2012b (link)), primary cultured hippocampal neurons were prepared from Sprague-Dawley rats. Pregnant dams at embryonic day 18 were sacrificed by asphyxiation with CO₂. After the fetuses were removed and decapitated, meninges-free hippocampi were isolated, trypsinized, and seeded onto 35-mm² poly-D-lysine coated glass-bottom tissue culture dishes. Neurons grown in Neurobasal™ medium containing L-glutamine, antibiotic/antimycotic and B₂₇ supplement were maintained in an incubator (37°C, 5% CO₂) for 10–14 days, at which time they were taken for experimentation. Neurons were treated with 12 different ART drugs for up to 48 hrs during which time the media was not changed. Measurements of endolysosomal pH, endolysosome sizes, and Aβ levels were conducted using separate dishes of cells. Typically, the purity of the neuronal cultures was more than 95% as determined by immunostaining of neurons with mouse anti-NeuN or goat anti-MAP2 antibodies (Millipore), and of astrocytes with mouse anti-GFAP antibody (Sigma).

Primary neuronal cultures were prepared from cerebral cortices of male and female Holtzman E17 rat embryos as described previously (Nash et al., 2019 ). Pregnant dams (embryonic day 17) were sacrificed by asphyxiation with CO₂. The fetuses were removed, decapitated, and meninges-free cerebral cortices were isolated, trypsinized, and plated onto 35-mm² poly-D-lysine-coated glass-bottom tissue culture dishes (Mattek, cat. no. P35GC-1.5–14-C). Neurons were grown in Neurobasal^™ medium (ThermoFisher, USA, A3582901) with L-glutamine, antibiotic/antimycotic, and B₂₇ supplement, and were maintained at 37°C and 5% CO₂ for 10–14 days at which time they were used for experimentation. Typically, the purity of the neuronal cultures was greater than 95% as determined by immunostaining with neuronal (mouse anti-NeuN or goat anti-MAP2 antibodies, Millipore, cat. no. AB15452 and MAB377) and astrocyte (mouse anti-GFAP antibody, Sigma, cat. no. G6171) markers. A total number of four pregnant dams and a total of 21 pups were used in this study to isolate neurons; the pups used were not separated by sex. Brains from the one mother were used for each separate culture.