Qcolor5

One day after the second dose of PDT, mice were sacrificed and samples of tumours with their adjacent SOTs and normal skins were excised, extended, sliced, fixed in 10% buffered formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by light microscopy under 4 ×, 10 × and 40 × magnifications (BX51 Olympus microscope, Japan), and photographs were documented with a digital camera (Olympus Q-color 5, Japan). The presence of tumour tissue, necrosis and other signals of tissue damage was evaluated. Epidermal and dermal damage, vascularity changes and the presence of lymphocytic infiltration were also investigated.

Linear dichroism measurements (n = 4) were obtained from toluidine blue-stained sections. Linear dichroism measurements have shown that GAG chains present in collagen fiber PGs are linearly distributed and predominantly parallel to the longest fiber axis [3 (link),39 (link),40 (link)]. In this case, linear dichroism is an extrinsic phenomenon, resulting from the arrangement of toluidine blue molecules that are electrostatically bound to the anionic link sites of the oriented substrate. The dichroic ratio (DR=dוו/d_┴) was determined by the toluidine blue absorbance in the parallel (dוו) and perpendicular (d_┴) positions of the tendon’s longest axis, with regard to the polarized light plane (PLP) [39 (link),40 (link)]. Linear dichroism was measured using an Olympus BX53 polarizing microscope (Objective: Olympus UPlanFL N 40×; Camera: Olympus Q-color 5; Polarizer: Olympus U-POT) and an image analyzer (Life Science Imaging Software, Version 510_UMA_-cellSens16_Han_en_00). The number of measurements (~100) of GA was represented as the median and they were chosen at random in 16 sections from four tendons of each group.

To induce small intestinal lesions, non-fasted animals were given a single subcutaneous injection of indomethacin at a dose of 15 mg/kg dissolved in 100% ethanol at 100 ul. Animals were then randomly assigned to receive either intraperitoneal injection of ZINC40099027 at 900 ug/kg or DMSO vehicle control every six hours for three days after allowing 24 hours for ulcer induction. At day 4, mice were anesthetized with isoflurane, blood was drawn by cardiac puncture for assay of serum levels of creatinine, ALT, and ZINC40099027, and animals were sacrificed by cervical dislocation before removing the small bowel for measurement of ulcers. In addition, small bowel ulcer tissue, liver, and kidney were saved in some animals for histological examination. The full length of the small intestine was excised at day 4 at sacrifice. The small intestine was gently washed with phosphate buffered saline, fixed in neutral-buffered 10% formalin for 5 minutes, and then opened along the anti-mesenteric attachment for imaging. Mucosal ulcers were imaged with an OLYMPUS Q Color 5 digital camera. Ulcer area was measured with Image J software and we summed the total area of all ulcers within each animal’s small bowel to achieve a final total ulcer area. Area measurements were made by an observer blinded to animal group assignments.