Rabbit anti hsp70

The NSCs were separated into seven groups as the coculture protocols. The samples were rinsed with PBS and fixed with 2.5% glutaraldehyde (diluted in 0.1 mmol/L PBS, PH 7.2~7.4). After that, the sections were dehydrated with the different concentration ethanol (70%, 80%, 90%, 100%, and 100% for every five minutes) and displaced by isoamyl acetate. Pictures were captured under scanning electron microscope (SEM; Carl Zeiss, Germany) after conventional critical point drying and gold-plating.
Nervous cells in coculture system (NSCs, neurons and glia) were, respectively, cultured in 12-well plates as the coculture protocols. Samples were fixed with 4% PFA, blocked by goat serum which was diluted into PBST, and immunostained with rabbit anti-HSP70 (1 : 100, Cell Signaling, Boston, MA, USA) and mouse anti-synaptophysin (1 : 500; Millipore, MA, USA) for 24 hours in the dark at 4°C. Sections were then followed by incubation with secondary antibodies (Fluorescein (FITC) AffiniPure goat anti-rabbit IgG (1 : 250, Jackson, PA, USA); DyLight 594 AffiniPure goat anti-mouse IgG (1 : 250; Jackson, PA, USA)). Pictures were captured under fluorescence microscope with 40x objective.

Lyophilized human Aβ_1–42 purified by HPLC was purchased from GL Biochem (Shanghai, China). We bought the rabbit anti-Hsp70, anti-Grp78, anti-caspase-12, anti-caspase-3, anti-p-Akt, and mouse anti-β-actin from Cell Signaling Technology (MA, USA) and rabbit anti-Akt1 from Millipore (MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (CA, USA), and annexin V-FITC/PI Apoptosis Detection Kit was purchased from Beyotime (Shanghai, China).

Cell extracts were prepared using a triton X-100 lysis buffer and the soluble fraction was isolated as previously described [10 (link)–12 (link), 58 (link)]. A Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) was used to determine the protein concentration and a total of 40ug soluble extract was added to each lane of a 4–12% gradient Bis-Tris polyacylamide gel and run in MES buffer (Life Technologies, Carlsbad, CA). The gel was then transferred overnight to a 0.1 uM nitrocellulose membrane and processed as previously described [14 (link), 58 (link)]. Primary rabbit anti-Hsp90 alpha, beta, or pan at 1:500 as indicated (Enzo Life Sciences, Farmingdale, NY), rabbit-anti-Hsp70 1:1000, mouse anti-Hsp27 1:1000, or rabbit anti-Hsp27 pS85 1:1000 (Cell Signaling Technology, Danvers, MA), mouse anti-tubulin 1:2000 (Abcam, Cambridge, MA), and secondary goat anti-rabbit IRDye 680CW and goat anti mouse IRDye 800CW 1:10, 000 (Licor, Lincoln, NE) were use according the manufactures directions. Images were collected using a Licor Odyssey system. Signal intensities were measured using ImageJ software (http://imagej.nih.gov/ij/). and analyzed using statistical analysis software as outlined below.