Cells have been vsualzed by confocal mcroscopy.All sectons of subcortcal whte matter analyzed contaned the corpus callosum, cngulum and external capsule, and were rostral of thehppocampus.Westerblot analyss For Westerblot analyss of whte matter lysates the subcortcal whte matter was dssected from 400um thck sectons prepared from CD1 mce thathad beereared hypoxa or normoxa.Brefly, brans have been slced coronally and only slces rostral of thehppocampus were utilised.Usng Roboz a fne straght and fne angled mcro dssectng forceps beneath a dssectng mcroscope the cortex was dssected away leavng the underlyng sub cortcal whte matter attached towards the stratum.The whte matter was theeasy pushed away from the stratum, leavng a thrbboof prmary whte matter tssue.The dssected whte matter was rnsed wth ce cold PBS thelysed oce 200 300ul of RPA lyss buffer.For vtro experments, cells were cultured 6 very well plates to approxmately 80 90% confluency and 1uM JAK nhbtor was additional on the cultures for 24hr or they were cultured hypoxc condtons for that ndcated tme perod.
The cells were washed twce wth ce cold PBS thelysed wth 250ul RPA lyss buffer for 30moce.Proteconcentratons have been determned by usng the Bradford proteassay kt.Westerblot analyss was performed o10 40ug of complete cell lysates.Protens had been resolved o4 20% Trs Glycne gels and transferred to great post to read mmoboPVDF membranes by tank blottng transfer buffer methanol, 8.3for 16hr at 4 C.The membranes had been thewashed Trs buffered salne wth 0.1% Twee20, ncubated for 1hr TBST contanng 5% bovne serum albumthencubated for 16hr at 4 C wth prmary antbodes duted TBST BSA.The membranes had been thewashed TBST three tmes for 10 mat room temperature followed by the addtoof etherhorseradsh peroxdase conjugated goat polyclonal ant rabbt gG for polyclonal prmary antbodes, orhorseradsh peroxdase conjugated goat ant mouse for mouse monoclonal prmary antbodes duted TBST BSA.The chemumnescent sgnals had been detected usng Perce ECL Westerblottng substrate.
X ray fms have been scanned usng aAgfa T1200 scanner and denstometrc measurements have been obtaned usng mageJ softwarlosome synaptosome D aspartate uptake assay and D aspartate uptake assay prmary astrocytes The glosome synaptosome uptake assays had been performed usng a modfed this article method of Weller Brans were eliminated on the gvetme pont afterhypoxc or normoxc rearng along with the whte matter was carefully dssected out.The tssue was thehomogenzed oce usng a mechancalhomogenzer tssue buffer and centrfuged at 14,000 g for 10 mn.The pellet was resuspended 250ul of sodum contanng Krebs buffer or sodum free Krebs buffer.Wheusng prmary astrocytes, 25,000 cells cm2 were cultured opoly L lysne coated 24 well plates.Cells have been taken care of wth 1uM JAK nhbtor or DMSO for 24hr, and thewashed twce wth warm Krebs buffer followed by addtoof 250ul

Krebs buffer.