Dmi 3000b light microscope

25 µl of 100% Matrigel (Fisher, catalog# 354234) was spread onto each well of an 8-chambered slide with precooled 200 µl sterile pipette tips. The plate was then placed in a 37 °C incubator for 30 min for the Matrigel to set. In the interim, cells were trypsinized, washed once with PBS, filtered through a 40 µm cell strainer to create a single-cell suspension, and counted. Cells were seeded on the Matrigel-coated slide at a seeding density of 5000 cells/well in a 300 µl volume of growth media containing 2% Matrigel. For each cell line or treatment condition, cells were seeded in triplicate and incubated at 37 °C for 7 days to allow for the growth of 3D cysts in suspension. During this time, the wells were supplemented with growth medium 72 h after initial placement into Matrigel. On day 7, the chamber slides were imaged on a Leica DMI 3000B light microscope. The images were analyzed using ImageJ software to obtain cyst size measurements. Each assay was repeated at least three times. Measurements from each experiment were combined and analyzed for statistical significance.

In the 12^th week, following a 12 h fast, blood samples were extracted via tail vein puncturing. Fasting blood glucose (FBG) was detected by a blood glucose meter (ACCU-CHEK® Performa; Roche Co., Ltd., Shanghai, China). For each rat, body weight was examined weekly after treatment. Also, the rats were singly housed in metabolic cages to investigate the food/water intake and urine volume. After that, animals were sacrificed by bleeding from the heart under pentobarbital sodium (45 mg/kg, i.p.) anesthesia, and then blood samples were centrifuged at 3000 rpm/min for 15 minutes at 4°C to collect the sera for the measurement of BUN, CHOL, LDL-C, AST, ALT, and ALB by a fully automatic biochemical analyzer (7020, Hitachi, Japan). The liver tissues were excised and weighed, and the hepatic index (liver weight/body weight ratio) was calculated. Subsequently, the partial liver was frozen at −80°C until further analysis, and the remnant liver was fixed in 4% paraformaldehyde.
For histological examination, the paraformaldehyde-fixed livers were embedded in paraffin and cut into 4 μm sections; the sectioned liver samples were then stained with hematoxylin and eosin. The images were captured and viewed at magnifications of 400x using a DMI3000B light microscope (Leica, Wetzlar, Germany).

The ability of cancer cells to form colonies was characterized using a soft agar assay. This assay required 21 days of growth on the soft agar medium. At the end of 3 weeks, a number of colonies formed per petri dish were counted using a crystal violet stain. Briefly, 1% sterile agar solution was warmed in a microwave and place to 37 °C water bath to cool down. 500 μg of agarose powder (BP165-25, Thermo Fisher Scientific, Fair Lawn, New Jersey, USA) was dissolved in 50 mL distilled water. The bottom of the petri dish (502014-07P, Sterilin petri dishes 9.6 cm², Dynalon Labware, Rochester, NY, USA) was coated with 0.7% agar and 0.3% CM by adding 3 ml/dish at room temperature for 30 min. following this, the upper layer of 3 mL of agar solution with 0.3% agar and 0.7% cell suspension (3125 cells/cm²) was plated. The top agar layer was allowed to solidify and then incubated for 3 weeks at 37 °C in 5% CO₂. CM was refreshed 2 times a week. In 21 days crystal violet was used as a staining for colonies (C3886, Sigma-Aldrich, Munich, Germany) and counting was performed using Leica DMI3000 B light microscope.