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For each mouse with each staining profile, two to three sections in the middle portion of the ARC were selected and images were collected; both sides of the ARC were manually outlined with an area of 0.03 mm on each side.
In order to measure the intensity of AT1 expression in dopamine neurons, all TH- immunopositive neurons (FITC fluorescence) within the nigrosome 1 AOI and within the matrix AOI in each photomicrograph had their perimeter manually outlined with an area-polygon selection tool in Nikon software NIS-Elements BR3 software (Nikon).
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The perimeter of each colony or fragment was manually outlined in every picture with Photoshop CS4.
In brief, the tumours were manually outlined using T2 data with freehand selection tool or the polygon selection tool.
Automated methods using atlases defined on brain templates are used as well as VOIs being manually outlined, particularly in cases with high levels of brain atrophy.
Therefore, for the subsequent analysis, the region of volume measurement (both feet) was manually outlined by each reader, with no attempts to manually remove apparent artefacts within the region of interest.
Automated image registration eliminates the subjective manual slice-matching and manually outlined ROIs are required only once.
The CA1 area in each image was manually outlined and the positive somata were removed by manual editing.
For TBI contusion volume assessment, the ipsilateral and contralateral hemispheres were manually outlined.
The alveoli were identified in the frames and manually outlined using Photoshop CS6 (Adobe Inc).
A VOI defining each organ insert was manually outlined using the Hawkeye CT data and transferred to the SPECT image.
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