Tissue from the ischemic area of the ipsilateral hemisphere from both vehicle-control and L-4F-treated T2DM stroke mice were isolated at 21 days after dMCAo. Brain-tissue lysate was subjected to WB analysis, as described previously (46 (link), 64 (link)). The following primary antibodies were used: AQP4 (1:5,000, Milipore, cat# ab3594), myelin basic protein (MBP, 1:500, Millipore, cat# MAB386), CD68 (ED-1, a marker of M1-macrophages, 1:1,000, Serotec, cat# MCA341R), monocyte chemoattractant protein-1 (MCP-1, 1:1,000, Abcam, cat# ab7202), toll-like receptor 4 (TLR4, 1:500, Santa Cruz, cat# sc-10741), Insulin-like growth factor 1 (IGF-1, 1:500, Santa Cruz, cat# sc-9013), IGF-1 receptor β (TGF-1Rβ,1:500, Santa Cruz, cat# sc-713), and β-actin (1:10,000, Abcam, cat# ab6276).
Total RNA was isolated using a standard protocol and quantitative RT-PCR was performed on an ABI Prism 7,000 Sequence Detection System using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). For each sample, the cDNA was generated and used to amplify GAPDH, AQP4, MBP, ED1, MCP-1, TLR4, IGF1, and IGF-1R as described previously (46 (link)). All the primers for the RT-PCR assay were designed using Primer Express software (ABI). Each sample was detected in triplicates.
Total RNA was isolated using a standard protocol and quantitative RT-PCR was performed on an ABI Prism 7,000 Sequence Detection System using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). For each sample, the cDNA was generated and used to amplify GAPDH, AQP4, MBP, ED1, MCP-1, TLR4, IGF1, and IGF-1R as described previously (46 (link)). All the primers for the RT-PCR assay were designed using Primer Express software (ABI). Each sample was detected in triplicates.
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