Computational protocol: Combination of Constraint-Induced Movement Therapy with Electroacupuncture Improves Functional Recovery following Neonatal Hypoxic-Ischemic Brain Injury in Rats

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Protocol publication

[…] All sections (20 μm) were stored in a storage buffer (30% ethylene glycol and 0.01% polyvinylpyrrolidone phosphate-pH 7.4) and were permeabilized for 30 minutes with PBS containing 0.3% Triton X-100. The sections were then incubated in a blocking solution of PBS-T (0.1% tween-20 in PBS) with 10% normal goat serum and were then exposed to the following specific primary antibodies: NeuN (1 : 200, Abcam ab177487, United Kingdom), GFAP (1 : 200, Millipore MAB360, Billerica, MA), Iba-1 (1 : 200, Wako 019–19741, Japan), and cleaved caspase-3 (1 : 200, cell signaling technology 9661L, Beverly, MA). Sections were then incubated with Alexa fluor 488- (1 : 500, Thermo fisher A-11001 and A-11008, Waltham, MA) or Alexa fluor 594-conjugated secondary antibodies (1 : 500, Thermo fisher A-11005 and A-11037) for 2 hours in the dark. We focused on the slices locating at 1,21 and −3.60 mm from the bregma and dashed red square in the cortex indicates the photographed area (Supplementary ). The fluorescence images were captured with a Zeiss LSM 700 laser scanning confocal device (Carl Zeiss, Jena, Germany), and image analysis was performed using ImageJ software (NIH, Bethesda, MD). [...] Data are expressed as mean ± standard error (SEM). The differences between the sham and HI groups were evaluated using unpaired t-tests. Comparison between the HI and treatment groups was made using repeated measurement one- or two-way ANOVAs followed by Tukey's post hoc tests. A P value of less than 0.05 was considered statistically significant. All statistical analyses were performed using SigmaPlot 11.2 (Systat Software Inc., San Jose, CA). […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus, Mus musculus
Diseases Brain Ischemia, Cerebral Palsy
Chemicals Oxygen