Image pro plus analysis system

Spinal cord tissue (1 mm above and below the lesion site, ~ 2 mm length) from the lesion site (T8–T9) was homogenized in RIPA buffer supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). Tissue lysates were subjected to western blotting using an anti-Arg-1 (Abcam, Cambridge, MA) or anti-Hpx (Abcam, Cambridge, MA) antibody. Protein bands were analyzed and quantified using densitometry and an Image-Pro Plus analysis system (Media Cybernetics, Silver Spring, MD) by normalizing their levels to the levels of GAPDH bands.

For hematoxylin and eosin (H&E) staining, organs were fixed in 10% phosphate-buffered formalin for 1 day and processed in a routine manner for paraffin sections. Tissue sections (5 μm) were cut and stained with H&E for microscopic examination. To quantify the size of epididymal and inguinal adipocytes, the H&E-stained sections were analyzed using the Image-Pro Plus analysis system (Media cybernetics, Bethesda, MD). Blood vessel staining was performed using a blood vessel staining kit (Chemicon, Billerica, MA, USA). Paraffin sections (3-μm thick) of adipose tissues were incubated with a rabbit anti-von Willebrand Factor (vWF) antibody as a primary antibody, goat anti-rabbit antibody as a biotinylated secondary antibody and streptavidin-alkaline phosphatase solution. A freshly prepared chromogen reagent was added to sections for the visualization of blood vessel. Blood vessel density was determined by Image-Pro Plus analysis system and blood vessel density was normalized with the number of adipocytes.

Histological observation was performed, and the percentages of TUNEL-positive, cleaved caspase-3-positive, and cleaved caspase-9-positive cells in each colonic tissue slice were measured by an Image-Pro^® plus computer-assisted image analysis system (Media Cybernetics Inc., Silver Spring, MD, USA) with a light microscope (Olympus BX51, Tokyo, Japan). To identify TUNEL-positive, cleaved caspase-3-positive, and cleaved caspase-9-positive cells, five visual fields in the colonic mucosa were randomly selected from each sample. At least 100 cells per field were observed under 200× magnification. The percentages of TUNEL-positive, cleaved caspase-3-positive, and cleaved caspase-9-positive cells were calculated using the following formula: positive cells/total cells × 100 (%). For the comparison of the relative expressions of proteins, the detected bands were quantified by a computer-assisted Image-Pro^® Plus analysis system (Media Cybernetics, Inc., Waltham, MA, USA). For the quantification, the value for the control group was set to 1.00.
Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Duncan’s post-hoc test using IBM SPSS Statistics software (ver. 23.0, IBM Co., Armonk, NY, USA). Significance was set at p < 0.05, and all experimental results were expressed as the mean ± standard error of the mean.