Sigma scan pro 5

The individual performing infarct volume analysis was not blinded to genotype but was blinded to the treatment groups. Mice were euthanized and brains collected at 96 h of reperfusion for 2,3,5-triphenyltetrazolium chloride histology and then digital image analysis of infarct volume was undertaken as previously published (Chen et al., 2012 (link)). Images were analyzed using SigmaScan Pro 5.0 (Systat Software, Inc., Point Richmond, CA, USA). To control for edema, regional infarct volume (cortex, striatum, and hemisphere) was determined by subtraction of the ipsilateral non-infarcted regional volume from the contralateral regional volume. This value was then divided by the contralateral regional volume and multiplied by 100 to yield regional infarct volume as a percentage of the contralateral region.

Net photosynthetic (Pn) and transpiration (E) rates were measured at the end of the drought treatment in six plants per treatment combination, and their weights added to the remaining leaves for fresh and dry weight determinations (n = 6) using the LI-6400XT portable open-flow photosynthesis system equipped with a red/blue LED light source (LI-COR, Inc., Lincoln, NE, USA). The PPFD was set at 400 μmol∙m⁻²∙s⁻¹, leaf temperature at 28 °C, and the reference CO₂ concentration was maintained at 400 μmol∙mol⁻¹ using the 6400-01 CO₂ mixer. All measurements were carried out between 4 and 8 h after the onset of the photoperiod on the upper fully expanded leaves. Leaf areas were calculated following computer scanning using the Sigmascan Pro 5.0 computer software (Systat Software, San Jose, CA, USA).

The extent of striatal dopaminergic denervation was measured by optical densitometry in four TH-immunostained sections from each animal, as previously described [59 (link), 62 (link)]. Sections were scanned in an Epson Perfection V750 PRO scanner, and the grey intensity of the staining in the striatum of both hemispheres of each section was measured using SigmaScan Pro 5.0 software (Systat Software, USA). The measured values were corrected for non-specific background staining by subtracting values obtained from the cortex. The optical density (OD) was calculated with the formula OD = −log (intensity in the striatum / intensity in the cortex).

Prepared PEG-AuNPs were drained and dried on copper grids for TEM measurement (H7650 Hitachi, Tokyo, Japan). Both AuNPs and PEG-AuNPs were counted using SigmaScan Pro 5.0 (Systat software, San Jose, CA, USA) and data from at least 300 nanoparticles was averaged for determining the particle sizes. The left lobe of mouse liver was harvested and fixed overnight in 2% paraformaldehyde and 2% glutaraldehyde. After the liver tissue was washed with PBS, samples were postfixed for 1 h in 2% OsO₄, then washed in ddH₂O, and finally dehydrated stepwise in increasing concentrations of ethanol. Resin embedding, sectioning to 100 nm slices by ultramicrotome, and TEM imaging and measurements were contracted to Bio MA-Tek Taiwan, which used a Hitachi HT7700 TEM system.

The measurements of Pn and E were carried out after 3, 6, and 9 days of treatments from approximately 5 to 9 h following the onset of photoperiod and by alternating plants from the different treatments. Three fully expanded uppermost leaves from each plant were marked and used for the measurements using a LI-6400 portable photosynthesis system with a 2 × 3 cm² leaf chamber (LI-COR Biosciences, Lincoln, NB, USA). The reference CO₂ concentration was 400 μmol mol⁻¹, the flow rate was 200 μmol s⁻¹, and the relative humidity (RH) level was set to 50% in the cuvette. The leaf chamber temperature was maintained at 20 °C, and PPFD was 400 μmol m⁻² s⁻¹ provided by the red-blue light spectrum of the light attachment. To determine leaf areas, the parts of the leaves that were inserted into the leaf chamber were excised after the last measurement and scanned. The leaf areas were calculated using the Sigmascan Pro 5.0 computer software (Systat Software, San Jose, CA, USA).

The measurement of the activity of the SOD isoforms and CAT was performed by electrophoresis in 10% polyacrylamide native gels. CAT and SOD isoforms were revealed according to the methods that were previously described by Pérez-Torres et al. [23 (link)]. Purified SOD from bovine erythrocytes with a specific activity of 112 U/mg of protein (Sigma-Aldrich, St. Louis, MO, USA) and purified CAT from a bovine liver having a specific activity of 60 U/mg (Sigma-Aldrich) were used as positive controls. The previously mentioned antioxidant enzyme activity determinations were performed according to the manufacturer's instructions. Samples were placed in a separate lane of the gel and run in parallel with the biological samples. The intensity of the signal from the controls was used as a reference to measure the enzymatic activity in the tissue samples. Therefore, the results are expressed as U of activity per mg of protein. The gels were analyzed by densitometry with image SigmaScan Pro 5.1 software (Systat Software, Inc., San Jose, CA, USA).