Biozero bz 8000k

Liver tissue samples from rats described above for Western Blot and Immunohistochemistry from the right hepatic lobe were carefully excised and stained using hematoxylin and eosin (HE).
Morphometric analyses of arterial vessels from the entire sample were performed using video microscopy (Keyence Deutschland GmbH, Germany, Biozero BZ-8000K). The image was captured and displayed on a computer monitor using an image analysis program (Keyence Deutschland GmbH, Germany, BZ image analysis application). The perimeter of the vessel lumen was measured in every vessel and taken as the circumference (C) of a circle. Lumen-Diameter (LD) was determined from the equation LD = C/π assuming that the cross section of the vessel was circular in vivo [10 (link)]. Wall thickness (WT) was measured eight times in every vessel (i.e. every 45°) as the linear distance between endothelium and adventitia and the values were averaged. Hematoxylin positive nuclei (N) were counted in each vessel. In order to compare different vessels, the ratio of wall thickness to diameter (WT/LD) and number of nuclei to diameter (N/LD) were calculated and used for comparison between the different groups [10 (link)].

The GUS histochemical assay of wildtype and hygromycin-resistant Euglena cells was performed as follows. The cells were collected by centrifugation at 3000× g for 5 min and washed with 0.5 mL of 200 mM phosphate buffer (pH 7) containing 4 mM EDTA. After washing, the cells were suspended in 0.5 mL GUS staining solution (50 mM NaH₂PO₄, 50 mM Na₂HPO₄, 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 3 mg X-Gluc (5-bromo-chloro-indolyl glucuronide cyclohexylammonium salt, Sigma-Aldrich Co., St. Louis, LA, USA), 5 mL Triton X-100, pH 7.0) [48 (link)]. The suspended cells were incubated overnight at 37 °C in the dark. After incubation, the cells were collected by centrifugation at 3000× g for 5 min to remove the staining solution and subsequently washed twice with 0.5 mL of 70% ethanol. Following washing, the cells were observed under a light microscope (Biozero BZ-8000K, Keyence, Osaka, Japan) at a 400× magnification. Blue staining was clear in transformants, but it was light green in the non-transformed controls.