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发布于:2021-6-4 00:09:41  访问:18 次 回复:0 篇
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Was presented as percent of such location in the total muscle
A lentiviral expression vector, pLenti6/Cdh5-R-Ras38V, was Title Loaded From File generated for EC-specific expression of Title Loaded From File R-Ras38V in mouse tissues. To confirm the induction of R-Ras38V expression, total tissue RNA was isolated in the GC muscle Title Loaded From File working with RNeasy Mini Kit (Qiagen, 74134). A set of mice that received pLenti6/Cdh5-R-Ras38V lentivirus injection into GC muscles had been treated with an Akt inhibitor MK-2206.Was presented as % of such location in the total muscle tissue location examined. For the analysis of muscle degeneration, five -thick cross-sections were obtained from mid-portion of GC muscle tissues and stained with hematoxylin and eosin. In addition, triphenyltetrazolium chloride (TTC) staining was utilized to assess the muscle viability55. Mice were killed at 14 days right after femoral artery ligation, as well as the GC muscle tissues were collected and reduce into 2-mm slices. These slices had been incubated for 30 min in two TTC (Sigma) resolution and fixed for 30 min in 10 (vol/vol) buffered formaldehyde. Viable tissues are stained in red and infarct locations are unstained (pale yellow/white). Immunostaining of GC muscle tissues. Ischemic and contralateral GC muscle tissues have been collected from mouse hindlimbs, fixed in 2 paraformaldehyde, dehydrated, embedded in paraffin, and sectioned at 5 m thickness. Soon after deparaffinization, rehydration, and antigen retrieval (DAKO), sections have been blocked and incubated overnight with main antibodies for CD31 (BD Biosciences) and R-Ras (AbCam). Sections have been then washed in PBS with 0.1 Triton X-100 and incubated with Alexa Flour-conjugated secondary antibodies for 1 h at area temperature for fluorescence microscopy. For immunohistochemistry of R-Ras, sections have been stained with anti-R-Ras polyclonal antibodies AR43 (present from Dr. J. Reed) and peroxidase-conjugated secondary antibody (Vector) followed by staining with AEC kit (Life Technologies). For immunofluorescence analyses of frozen sections, GC muscles had been snap-frozen in liquid nitrogen. Muscle cross-sections ofNATURE COMMUNICATIONS | eight:| DOI: ten.1038/s41467-017-01865-x | www.nature.com/naturecommunicationsARTICLEEndothelial cell-targeted in vivo gene transfer. A lentiviral expression vector, pLenti6/Cdh5-R-Ras38V, was generated for EC-specific expression of R-Ras38V in mouse tissues. This construct was generated by replacing CMV promoter within the original pLenti6 vector with the mouse VE-cadherin (Cdh5) promoter sequence35. A similar expression vector was constructed for R-Ras38V D64A mutant expression. A lentiviral vector with Cdh5 promoter with out cDNA insert was also generated as a handle. For R-Ras KO rescue experiments, hindlimb ischemia was induced in the R-Ras KO mice, and 3 days later, mice had been randomly assigned into two groups. The lentivirus harboring the pLenti6/Cdh5-R-Ras38V expression vector or manage vector was intramuscularly injected in to the GC muscle at three injection web pages, 10 each, 1.two ?107 TU total making use of a Hamilton syringe. At day 14, mice had been intravenously perfused with biotinylated tomato lectin by way of the tail vein 5 min prior to resecting the GC muscle tissues for evaluation. Five mice per group had been examined. To confirm the induction of R-Ras38V expression, total tissue RNA was isolated from the GC muscle using RNeasy Mini Kit (Qiagen, 74134).
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