Metamorph 4

Renal scarring was studied using morphometry in previous studies [14 (link), 15 (link)]. Specifically, renal biopsy specimens that were 5 μm thick underwent Jones’ silver staining. Semi-quantitative computerized image analysis was then performed using the Leica Twin Pro image analysis system from Leica Microsystems in Wetzlar, Germany. The analysis utilized MetaMorph 4.0 image-analyzing software from Universal Imaging Corporation in Downingtown, PA, USA. Each patient’s sample included ten glomeruli and ten randomly selected areas, which were evaluated to determine the average percentage of scarred glomerular and tubulointerstitial areas.

Jones’ silver staining was performed on 5 µm thick sections of the renal biopsy specimens of each patient. As previously described by others [34 (link)], we used a computerized image analysis method to semi-quantify nephrosclerosis. Briefly, a Leica Twin Pro image analysis system (Leica Microsystems, Wetzlar, Germany) was connected to a Leica DC500 digital camera on a Leica DMRXA2 microscope working with a 40× objective (final calibration: 0.258 mm/pixel), which was connected to a microcomputer for storage of the morphometric measurements so that image analysis could be performed using image analysis software (MetaMorph 4.0; Universal Imaging Corporation TM, Downingtown, PA, USA). A total of 10 glomeruli and 10 randomly selected areas were assessed in each patient and the average percentage of scarred glomerular and tubulointerstitial areas, as represented by the percentage of the area with positive staining, was computed for each patient.

Histological lesions were classified by the revised Oxford classification [19 (link)]. The severity of glomerulosclerosis and tubulointerstitial fibrosis were further assessed by morphometric study as described previously [20 (link), 21 (link)]. Briefly, Jones’ silver staining was performed on 4 μm thick sections of renal biopsy specimen. Computerized image analysis method was then performed by the MetaMorph 4.0 image-analyzing software (Universal Imaging Corporation™, Downingtown, PA). Ten glomeruli and 10 randomly selected tubulointerstitial areas were assessed for each patient.

To compare the changes of Trx2 and Prx3 in the hippocampal CA1 region between adult and aged gerbils, immunohistochemistry for rabbit anti-Trx2 (1:500; cat. no. LF-PA0024, Ab Frontier, Seoul, Korea) and mouse anti-Prx3 (1:500; cat. no. LF-MA0045; Ab Frontier) was performed, according to the above-mentioned method. In order to establish the specificity of the immunostaining, a negative control was used, with only the secondary antibody and without primary antibody. This negative control resulted in the absence of immunoreactivity in any structures.
A total of six sections with a 120 μm interval per animal were selected to quantitatively analyze the Trx2 and Prx3 immunoreactivity. Digital images of the hippocampal CA1 region were captured using an AxioM1 light microscope (Carl ZeissAG), equipped with a digital camera (Axiocam; Carl Zeiss AG) connected to a PC monitor. According to the methods of our previous study (7 (link)), semi-quantification of the immunostaining intensities were evaluated using digital image analysis software (MetaMorph 4.01; Universal Imaging Corporation, Downingtown, PA, USA). The level of immunoreactivity was scaled as −, ±, +, ++ or +++ representing no staining (gray scale value ≥200), weakly positive (gray scale value=150–199), moderate (gray scale value=100–149), marked (gray scale value=50–99), or very marked (gray scale value ≤49), respectively.