The Cell lysates were separated on SDS gel according to the manufac turers protocols. The proteins were then transferred onto PVDF and blotted with various antibodies as indicated in the figures. Protein bands were detected by chemiluminescence www.selleckchem.com/products/mek162.html reagents. Immunofluorescent staining Cells were seeded on a poly D lysine coated cover slip of 12 mm diameter at a density of 1 �� 105 in 24 well plate. After chemical hypoxia, cells were fixed with 4% para formaldehyde for 30 min, permeabilized with 0. 1% Triton X 100 for 10 min, and blocked with 2%BSA in PBS for 30 min. Cells were stained with primary antibodies for 1 hour. After washing, cells were incubated with corre sponding Alexa Fluor 488 594 conjugated secondary antibodies for 1 hour.
Stained cells were mounted with SlowFade gold antifade reagent with DAPI, sealed with nail polish, and observed with a confocal laser scanning microscope. Negative controls were obtained by substitut ing the primary antibodies with corresponding immunoglobulin isotypes. Evaluation of cell viability MTT reduction assay Cell Inhibitors,Modulators,Libraries viability was determined by 3 2, 5 diphenyl tetrazolium bromide assay. Cortical cells were seeded with density of 2 �� 104 in a 96 well plate then cultured for 12 days. 10 uL of MTT stock solution was added to the hypoxic cells and incubated 2 hrs at 37 C. The resulting MTT formazan was extracted with 100 uL of detergent. The Inhibitors,Modulators,Libraries reaction product was analyzed at 570 nm with a microplate spectrophotometer. TUNEL assay Apoptotic cells were determined by terminal deoxynucle otidyl transferase dUTP nick end labeling assay.
TUNEL staining was performed according to the manufacturers protocol. Cells were fixed with 4% para formaldehyde. Apoptotic cells were identified by strepta vidin fluorescein detection of biotinylated dUTP incorporation. Stained slides were visualized by confocal Inhibitors,Modulators,Libraries laser scanning microscopy. Positive cells were counted and considered as apoptotic cells. Evaluation of cellular function Electrophysiological recordings Whole cell recordings were made from 14 18 days cul tured cortical neurons at room temperature. Cover slips containing cells were transferred to a small stage mounted on an inverted microscope and were superfused with extracellular saline solution containing, 137 Na Isethionic acid, 4 K gluconate, 1. 8 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose.
Recording electrode pipettes had resis tances of 2 4 Inhibitors,Modulators,Libraries M and were filled with an internal pipette solution containing, 130 K gluconate, 1 MgCl2, 5 EGTA, 5 MgATP, 10 HEPES and 0. 4 Na2GTP. Action potentials were evoked by injecting depolarization Inhibitors,Modulators,Libraries current into primary cortical neurons under normal conditions and recovery after treatment with 1. http://www.selleckchem.com/products/BI6727-Volasertib.html 5 mM NaCN for 30 mins. Recordings were not performed during NaCN treat ment because the Ag AgCl reference electrode can be oxidized by NaCN which causes a pseudo neuronal mem brane depolarization artifact. Data were collected via a patch clamp amplifier, stored on a PC, and analyzed by pClamp 9.