Pas reagent

Sections were stained with periodic acid-Schiff (PAS) reagent (Sigma-Aldrich, St. Louis, MO). Three sections were examined for each cornea, and images were captured at equidistant intervals from the corneal mid-point with a DS-Fil digital camera (Nikon, Tokyo, Japan) attached to a Nikon microscope.

Glycogen staining was performed using the periodic acid–Schiff (PAS) reagent (Sigma-Aldrich Corp.). Cells were fixed in formalin–ethanol solution for 1 min at room temperature and stained with 0.5% Schiff's reagent for 15 min. After washing cells with PBS several times, stained cells were observed and photographed. Cells accumulating glycogen were quantified as the number of PAS-positive cells (magenta) per total cell count in the same microscopic field. The number was determined by counting at least five random fields per sample. To determine the origin of the staining, cells were treated with 0.5% amylase (Sigma-Aldrich Corp.) for 90 min at 37 °C to degrade intracellular glycogen before PAS staining.

After induction of hepatocytes with a hormone ratio (insulin/glucagon) gradient for the prescribed interval (24 hours), the microfluidic device was disconnected from the syringe pumps. Cells were, then, fixed with 100% methanol for 20 min at −20 °C. The PDMS microfluidic device was peeled off from the glass microscope slide by cutting around the pattern of cell culture chamber of PDMS layer on the surface of glass microscope slide with a razor blade before further analysis. Cells were treated with PAS reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instruction to assess the level of glycogen in the primary hepatocytes as a marker of carbohydrate metabolism in hepatocytes. Images were captured on a Nikon microscope (Diaphot TMD, Nikon, Tokyo, Japan) at a 10X magnification with an attached Nikon D5100 digital SLR camera.

Kidneys were fixed in Bouin’s solution (Sigma-Aldrich, Dorset, UK) for 2-4 hours and then transferred to 70% ethanol. Sections were embedded in paraffin and stained with periodic acid-Schiff (PAS) reagent (Sigma-Aldrich, Dorset, UK) and examined by light microscopy. Slides were reviewed in a blinded fashion and a semi-quantitative analysis was undertaken with sections being scored for glomerular hypercellularity (0-4), segmental Sclerosis (0-1), and mesangial expansion (0-1). A minimum number of 10 glomeruli were scored per mouse and an average taken.

Kidneys were fixed with 10% formalin (Junsei Chemical, Tokyo, Japan), and embedded in paraffin. Cutting 5 μm slides were stained with periodic acid–Schiff (PAS) reagent (Sigma, MO, USA) and Picrosirius Red Stain Kit (PolySciences, PA, USA), respectively. All slide samples were reviewed and scored by the unaware pathologist. Histological changes and fibrosis were analyzed by using the EVOS M5000 imaging system (Thermo Fisher Scientific, Waltham, WA, USA). Areas of glomerular injury in each image was quantified using ImageJ software (NIH, Bethesda, MD, USA).

Paraffin-embedded kidneys were cut in 3 µm sections. Xylol was used for deparaffinisation and an ethanol gradient for dehydration. Sections were stained with periodic acid Schiff (PAS) reagent (P7875, Sigma-Aldrich) and counterstained with haematoxylin (1.09249, Sigma-Adrich and H02-1000, Dr. K. Hollborn & Söhne, 1:1 mixture). Stained slides were digitalised for morphometric analysis using an Axio Scan.Z1 slide scanner (40 × magnification) and ZEN 3.1 microscopy software (blue edition) (both Carl Zeiss Microscopy). To quantify histological changes of BTBR ob/ob compared to BTBR wt/wt mice, renal corpuscle area and glomerular tuft area of 20 randomly selected glomeruli per animal were measured using QuPath version 0.4.4^{101 (link)}. Bowman’s space area was calculated through subtraction of the glomerular tuft area from the renal corpuscle area. Mesangial matrix expansion was assessed by a pathologist with expertise in nephropathology (T.A.) in a blinded manner. To this end, the amount of mesangial matrix compared to the glomerular tuft area was analysed in 20 glomeruli of each animal, which were graded on a scale of 1 to 4, defined as follows: 1, 0–10%; 2, 10–50%; 3, > 50%, 4, glomerular sclerosis.