Western blot, MBP proteins were separated by sodium dodecyl sulfate polyacrylamide gel electropho resis and transferred onto a polyvi nylidene difluoride membranes. Phosphate buffered saline with Tween ten was applied to wash the gel movies 5 min by three occasions, then the movies had been additional rabbit anti rat MBP key antibodies to incubate 2 h and washed by PBST for ten min by 3 occasions, then incubated 1 h in horseradish peroxidase goat anti rabbit antibodies, lastly washed with PBST and PBS successively for 5 min by 3 times. The gel film photographs was deve loped inside a B mixed developing agent and scanned with Bio Rad 2000 gel imaging method to analysed gray value of strap by Quantity 1 software package. Within the similar specimen, the gray worth of B action, as an inner para meter, was also detected to calibrate the content material of every target protein.
The relative articles of protein the gray value of MBP the gray worth B action. inhibitor Epigenetic inhibitor The experi ment was repeated three occasions as well as the outcomes presented with indicate normal deviation. Reverse transcription polymerase chain response Extraction of total RNA, Chose 5 rats from control group and model group respectively and rats in therapy group randomly and anesthetized by chloral hydrate after treatment 24 h. Took 200 mg ischemic brain tissue and place into 1. 5 ml EP tube. Added RNA Solv reagent one ml, minced and grinded, oscillated ultra sonically for thirty s and placed 5 min at area temperature, and centrifuged for 15 min. Took the supernatant into another EP tube and extra chloroform 0. 2 ml, shocked and mixed 15 s, placed on ice for ten min and centrifuged for 15 min.
Then, collected supernatant into a further EP tube and join iso propyl alcohol 0. five ml, blended gently, then placed within the ice for 10 min, centrifuged for 15 min and discarded supernatant. Washed precipitation working with 1 ml 75% alcohol, mixed and centrifuged for 5 min, then abandoned supernatant thoroughly, dried kinase inhibitor OSI-906 30 min in fume hood, and put in 57 C water bath for ten min soon after incorporating 0. 1% DEPC H2O thirty ul. The purity and abundance of RNA have been established by ultraviolet spectrophotometer and stored at ?twenty C. RT PCR, Primers had been made with Premier 5. 0 computer software and synthesized by Shanghai Invitrogen Co. Ltd. Target gene NSE, sense primer, Reverse tran scription synthesis method of cDNA, Oligod T two ul and RNA two ug with DEPC H2O extra to 13. four ul, and the mixed liquid was placed at 70 C for 5 min, then ice bath for five min. Plused M MLV RT five × five ul, dNTP mixture 5 ul, RNase inhibitor 0. 62 ul and M MLV RTase one ul.