Rt2 profiler software

PCR array data were analyzed using RT2 Profiler software (Qiagen). The p values were calculated based on a Student’s t test of the replicate 2^(−Delta Ct) values for each gene in the control and inhibitor groups. Statistical analysis for all other obtained data was performed using Prism and GraphPad Prism v.6.05, or R software. Two-way ANOVA followed by Tukey’s multiple comparison test was used for cytokine protein expression analysis. Student’s t test was performed for the Western blot, immunofluorescence microscopy, and electron microscopy data analyses.

Mouse cytokine and chemokine PCR array (Qiagen, Cat# PAMM-150Z, http://www.sabiosciences.com/rt_pcr_product/HTML/PAMM-150A.html) was utilized to evaluate the expression of genes encoding major pro- and anti-inflammatory cytokines and chemokines in the RNA samples. At 7 days after dMCAO, three brains per experimental group (inhibitor and control) were used to generate separate sample triplicates for the analyses. Brain cortices were dissected and stored in RNAlater solution (Ambion). Total RNA was isolated using mirVana miRNA (AB/Ambion) isolation kit, from the hemispheres ipsilateral to dMCAO injury. All measurements and data quantification were performed by Qiagen Company Sample & Assay Technologies team. The obtained raw data were analyzed using RT2 Profiler software (Qiagen). Only the genes with consistent expression levels (within the triplicate samples) were picked up for statistical analysis. The fold changes of gene expressions (inhibitor vs control) were calculated, and the transcripts that showed ≥2-fold change in expression (either up- or downregulated) were retained. At the final step, statistical significance (p value <0.05) and reliability of the results was automatically evaluated. The raw data are deposited in the Open Science Framework general data repository, link: https://osf.io/3zhc4/?view_only=0826f6e687884b90ab774328c2746ae1.