55i microscope

The histopathological assessment was conducted by hematoxylin and eosin (H&E) staining following a previous study (Liu et al., 2020 (link)). Briefly, brain and colon tissues were collected, perfused with 4% paraformaldehyde phosphate buffer, and then dehydrated with ascending concentrations of ethanol (50%, 70%, 80%, 95%, and 100%, for 5 min each). The tissues were subsequently cleared with xylene, embedded in paraffin, and sectioned into 4 μm thickness. Each section was perfused in xylene (for 10 min twice) to remove the wax. The sectioned tissues were soaked in descending concentrations of ethanol (100%, 95%, 90%, 80%, 70%–0%, for 5 min each). The sections were stained with hematoxylin for 5 min, washed with running water for 1 min, soaked in an alcoholic solution of 1% hydrochloric acid for 3 s, rinsed with running water for 10 min, and stained with eosin for 3 min. This was followed by soaking in 95% ethanol (for 5 min twice), 100% ethanol (for 5 min twice), and xylene (for 2 min three times). Finally, the stained sections were sealed, mounted using Permount™ Mounting Medium, and imaged and analysed using a light microscope (Nikon 55i microscope, Nikon, Japan).

After blood was obtained from the mice, the intact livers were photographed and weighed. Liver tissue was placed in a solution of 4% paraformaldehyde and it was kept at −80°C. The liver was sectioned and the thickness was limited to 5 μm. Xylene and gradient alcohol was used for dewaxing, and hematoxylin and eosin were used for staining and sealed with neutral resin. Neutral resin was used to glue coverslips, which were observed and photographed using a Nikon 55i microscope (Nikon, Japan). The Non-Alcoholic Fatty Liver Activity Score (NAS) was applied in this drug treatment trial (Kleiner et al., 2005 (link)). In the NAS scoring system, hepatocellular steatosis was scored 0–3, hepatocellular ballooning was scored 0–2, and inflammation within the liver lobules was scored 0–3.

Kidneys were fixed in 10% buffered formalin, embedded in paraffin, cut into 4-μm sections and stained with hematoxylin and eosin (H&E) and/or Masson’s trichrome (n = 5–6 per group/time point). ARHGEF11 localization in human kidney biopsy was assessed by immunohistochemistry on unstained sections using primary antibodies directed at ARHGEF11 (Novus Biologicals, Co) and detected by DAB (Ultravision LPValue Detection System, Thermo Scientific). Slides were counterstained with methyl green. Images were captured using Nikon 55i microscope with DS-Fi1 5-Meg Color C digital camera (Nikon, Melville, NY).