Leica cm1860 cryostat

The FBG was determined using a blood glucose meter (Roche, Basel, Switzerland). Glycosylated hemoglobin (HbA1c) was detected with a Quo-Test HbA1c Analyzer (QUOTIENT Diagnostics Ltd. Walton-on-Thames, Surrey, UK). The urine creatinine and urea nitrogen were measured using an AU480 automatic chemistry analyzer (Beckman Coulter Inc., Brea, CA, USA).
The frozen kidney tissues were cut into 8 μm serial sections at −22 °C using a Leica CM1860 cryostat (Leica Microsystems Ltd., Wetzlar, Germany) and mounted onto adhesion microscope slides (Thermo Scientific, CA, USA). Hematoxylin and eosin (H&E) staining of kidney sections was performed to reveal the histopathological lesions.

SDH staining was carried out as described by Hadsell et al. (2011) (link). Frozen OCT blocks of liver and mammary tissue were cryosectioned between −15 and −20° C on the Leica CM1860 Cryostat (Leica Microsystems Inc., Buffalo Grove, IL, USA) at Purdue University Histology Research Laboratory, and 5-µm tissue slices were placed on glass slides (Hadsell et al., 2011 (link)). Frozen sections were incubated in a solution of 4 mg/ml nitro blue tetrazolium chloride, 0.2 M Tris, 0.05 M MgCl₂, and 0.83 M sodium succinate (Sigma-Aldrich) for 1 h at 37°C. The sections were then transferred to 15% formol saline containing 0.9% w/v NaCl and 15% w/v paraformaldehyde and incubated for 15 min at room temperature. Sections were washed in distilled water for 3 min, dehydrated in 93%, 95%, and 100% alcohol for 3 min each. The sections were rinsed in xylene, mounted with two drops of DPX mounting medium (Sigma Aldrich), and cover slipped.
Four images of liver or mammary tissue sections per mouse were captured using Nikon Eclipse 50i microscope (Nikon Inc., New York, NY, USA; Evolution MP, Media Cybernetics Inc., Rockville, MD, USA) microscope using the 40X objective. Percent area of staining in each image was measured using Image J software. Prior to measuring area, images were converted to black (stained area) and white (absence of stain) using the eight-bit threshold tool.

Liver, jejunum and adipose tissues were embedded in FSC 22 frozen section compound (Leica Biosystems, Richmond, IL, USA), then frozen and sectioned at 5 mm using a Leica CM1860 Cryostat (Leica Microsystems, Nussloch, Germany). Sections were then stained with oil red O solution or hematoxylin and eosin (Cayman chemical, USA), after which they were mounted on silicone-coated slides (Leica, USA) and examined using an Olympus BX61 microscope (Tokyo, Japan) and photographed using an Olympus DP70 digital camera (Tokyo, Japan).