Nis elements br

For microscopic analysis, 5 μL of exponential growing cells at N = 70 were transferred onto a microscope slide and analyzed using an Eclipse E600 (Nikon, Tokyo, Japan) microscope equipped with a 100 × NA 1.4 oil immersion objective and a DXM1200C camera (Nikon, Tokyo, Japan). The image analysis and determination of the cell size were performed with the Nikon Nis-Elements BR (version 3.2) imaging software or/and ImageJ program (version 1.50). Fluorescence signals of GFP were visualized utilizing the Epi-FL filter block for fluorescence B-2E/C (EX: 465–495, DM: 505, BA: 515–555; Nikon, Tokyo, Japan), thereby using identical fluorescence intensity and exposure times between 1 and 2 s.
Cell length of parental strain R6, C405, and R6_2xC405 growing in C+Y medium at 30 °C were measured from phase-contrast images by using the Nikon Nis-Elements BR software (version 3.2), and the results were further analyzed in Microsoft Excel. More than 100 cells from at least two independent experiments were measured for each strain. Only cells showing a clear invagination at the division septum (late divisional cells) were measured.

Histological evaluation of fibrosis was performed on 20 male mice belonging to 4 groups (5 per group): (1) three-month-old untreated, (2) six-month-old untreated, (3) twelve-month-old untreated, and (4) twelve-month-old treated. At necropsy, heart ventriculi were collected and referred to the histology laboratory. Samples were paraffin embedded and sectioned to show both ventricular walls. After rehydration of sections, Mallory trichrome staining was performed by using a commercial kit (04-020802, Mallory trichrome, Bio Optica, Italy); slides were thus dehydrated, cleared, and mounted with permanent mounting medium. Five images at 20x magnification were collected focusing at connective tissue hot spots and semiautomatically analyzed with the image processing software Nis-Elements Br (Nis-Elements Br, Nikon Instruments, Italy). Fibrosis was expressed as percent of the images occupied by the connective tissue (stained in blue with Mallory trichrome).
Data analysis. Values were analyzed by Bonferroni's multiple comparison test as post hoc analysis after one-way analysis of variance; significance was set at p < 0.05.

For light microscopic analysis, mouse kidneys were fixed with 4% buffered paraformaldehyde (pH 7.4) at 4°C overnight, dehydrated in graded alcohols, and embedded in paraffin. The paraffin-embedded kidneys were cut into 4-μm sections and stained with periodic acid-Schiff (PAS) for the evaluation of pathologic changes. Twenty five glomeruli and approximately 80–100 proximal tubules in each mouse and six mice in each group were measured for mesangial matrix fraction and tubular area using Nikon NIS-Elements BR software version 4.10.00 (Nikon, Japan) by a pathologist in a blinded manner.