Periodic acid schiff staining kit

Kidneys were fixed in 10% buffered neutral formalin, further processed and paraffin embedded. Three micrometer sections were cut and stained using a Periodic acid Schiff staining kit (Sigma) according to the manufacturers' instructions. Tissues were imaged using a Leica DN2000 microscope and micrographs taken using Leica Application Suite software. Image analysis was performed using ImageJ; all images were contrast enhanced using the same parameters.
Glomerulosclerosis was scored for each glomerulus as follows: 0 = normal glomeruli; 1 = up to 25% involvement; 2 = 25%‐50% involvement; 3 = 50%‐75% involvement; 4 = over 75% involvement. The glomerulosclerosis index was calculated according to the formula ([1 × N1] + [2 × N2] + [3 × N3] + [4 × N4])/(N0 + N1 + N2 + N3 + N4), where Nx is the number of glomeruli with each given score.

Testes were isolated and weighed followed by fixation in Bouin’s solution or 4% paraformaldehyde and parrafifin embedded. For analysis of testis morphology, Bouins fixed tissue sections (5 μm) were stained with a periodic acid-Schiff staining kit (Sigma, St. Louis, MO) followed by staining with hematoxylin. For analysis of EGFP or AR expression, paraformaldehyde fixed sections were probed with either primary goat antisera against GFP (Abcam Inc., Cambridge, MA ab5450, 1:1000 dilution) or polyclonal primary rabbit antisera against AR (Santa Cruz Biotechnology, Santa Cruz, CA AR C19, Sc-815, 1:1000 dilution). For EGFP imunodetection, biotin conjugated mouse anti-goat secondary antisera (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) was added and bound antibodies were detected as described by the kit instructions using DAB staining solution. Slides were counterstained with hematoxylin. For AR immunodetection, secondary donkey anti-rabbit antisera with conjugated cy3 fluor was added followed by staining with DAP1(4',6-diamidino-2-phenylindole). A charged coupled device (CCD) video camera system was used to capture images of tubule cross-sections.

To determine the secretion of human albumin and urea, HCC cells were seeded in 6-well plates for 24 h, and the medium was then replaced with fresh DMEM without fetal bovine serum for another 24 h. The supernatant was collected according to the manufacturer’s instructions. Human albumin was measured with a Human Albumin ELISA Quantitation kit (Assay Pro), and urea was measured with a QuantiChrom^TM Urea Assay Kit (BioAssay System). The Periodic-Acid Schiff staining kit was obtained from Sigma-Aldrich. Cells were fixed and stained following the manufacturer’s instructions. For oil red O staining, HCC cells were stained with oil red O (Sigma-Aldrich) according to the manufacturer’s instructions.

Formalin-fixed, paraffin-embedded renal tissues including ATN and normal control were sectioned at 4 µm thickness and stained for histological examination. These sections were stained with a periodic acid-Schiff staining kit (Merck Millipore, Billerica, MA, USA) and Masson’s trichrome Kit (American Master Tech Scientific, Lodi, CA, USA) to determine the severity of tubular injury and percentage of interstitial fibrosis, respectively. All sections were examined by a pathologist (T.-C.S.) unaware of the clinical and laboratory data. The characteristics of tubular injury included tubular cell swelling, loss of brush border, or nuclear condensation. The severity of tubular injury was scored from 0 to 4 according to the percentage of the injured area of the section (0—no change; 1—changes affecting 1–25%; 2—changes affecting 25–50%; 3—changes affecting 50–75%; 4—changes affecting 75–100% of the section).