High resolution scans were taken with an Epson flatbed scanner at 600 DPI. The level of staining intensity was measured from 4 striatal sections (AP + 1.6, + 0.7, –0.26, and –0.6 mm) for αSyn211-DAB, GFP-DAB and TH-DAB and one midbrain section (AP: –5.6 mm) for OX42-DAB, using imagej software (NIH, Version 1.8.0). Before measurement, each image was transformed into grayscale 8-bit and calibrated using a step-tablet from Epson with known OD values using the Rodbard function (imagej.nih.gov/ij/docs/examples/calibration/">https://imagej.nih.gov/ij/docs/examples/calibration/ ). Correction for non-specific background was done by subtracting values obtained from the corpus callosum to the measured values. The regions of interest were outlined, and the grey-pixel intensity value average was measured. The data are expressed as optical density values of contralateral vs ipsilateral striatum or midbrain.
Imagej
Manufactured by Epson
Sourced in United States
ImageJ is a free, open-source image processing software designed for scientific and research applications. It provides a wide range of tools for image analysis, processing, and quantification. ImageJ is capable of opening, displaying, editing, analyzing, processing, saving, and printing a variety of image file formats.
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4 protocols using imagej
1
Quantifying Striatal and Midbrain Protein Levels
2
Cresyl Violet Staining for Infarct Quantification
3
Native PAGE for SOD Activity
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Separations of soluble protein fractions were performed using native non-continuous PAGE in the buffer system described by Laemmli (1970) (link) at 4°C and 180 V. SOD bands on 12% polyacrylamide gels were visualized according to the staining procedure described by Beauchamp and Fridovich (1971) (link). The gels were incubated in the staining buffer for 30 min, in darkness, at room temperature and subsequently exposed to white light until SOD activity bands became visible. The gels were scanned using the office scanner Epson V700 Photo, and densitometric analysis was performed with ImageJ (NIH, United States).
4
Protein Expression Analysis by Western Blot
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For Western blotting, cells were lysed in Laemmli buffer (4% sodium dodecyl sulfate, 20% glycerol, 120 mM Tris-Cl, pH 6.8, and 0.02% bromophenol blue). Lysates were separated by SDS-PAGE gel and electrotransferred to a nitrocellulose membrane. Blots were blocked in PBS with 5% nonfat dry milk and incubated with primary antibodies at 1:1,000 overnight at 4°C. Blots were incubated in secondary antibodies at 1:10,000 for 1 h at room temperature and developed with ECL Western blotting substrate (Thermo Fisher Scientific). Blots were scanned as film negatives on a photo scanner (Perfection v700; Epson) and analyzed using the gel analysis tool in ImageJ (National Institutes of Health). The intensity of the protein bands was normalized through comparison with the loading control bands.
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