Ckx41 light microscope

Migration assay were carried out according to previous study [17 (link)]. In this experiment, MDA-MB-231 cells were seeded in six-well plates at 1 x 10⁷ cells/well. Straight lines were created using a sterile 200 μL pipette tip once the cells reached 100% confluency. The plate was rinsed with sterile PBS to remove floating cells. Cells were then exposed to 0–2 mg/mL CN extracts diluted in serum-free RPMI 1640 media. The wound conditions were monitored for 24 h. Images were captured using Olympus CKX41 light microscope at 20X magnification.

The liver and adipose tissues were fixed in 10% formalin solution, embedded in paraffin and sectioned at 5 µm of thickness. The sections were stained with hematoxylin and eosin (H&E) according to the standard protocols for histological analysis and all staining images were captured using a CKX41 light microscope (Olympus, Tokyo, Japan). The NAFLD activity score (NAS), established by the Pathology Committee of the Non-alcoholic steatohepatitis (NASH) Clinical Research Network [50 (link)], was used for hepatic pathological scoring; 3 histological features (steatosis, lobular inflammation, and hepatocellular ballooning) were evaluated semi-quantitatively. Steatosis was scored (0–3); 0 = 0–5%, 1 = 6–33%, 2 = 34–66%, and 3 = 67–100% of hepatocytes containing fat droplets. Lobular inflammation was scored (0–3); 0 = no foci, 1 = more than 2 foci per 200× field, 2 = 2–4 foci per 200× field, and 3 = greater than 4 foci per 200× field. Hepatocyte ballooning was scored (0–2); 0 = none, 1 = few, and 2 = many/prominent ballooned cells. The final NAS is the unweighted sum of the three scores of steatosis, inflammation, and ballooning; NAS ≥ 5 are diagnosed as “NASH” and NAS < 3 as “not NASH”.

Growth factor-reduced 8.0 micron Matrigel Invasion Chambers (Corning, Tewksbury MA, USA) were added to a 24-well plate. Matrigel invasion chambers were hydrated for 1 h at 37°C with 250 μL of McCoy’s 5A medium containing 0.2% FBS and penicillin-streptomycin. T24 cells were then seeded in McCoy’s 5A medium without FBS at a concentration of 150,000 cells/mL. Following hydration, 250 μL of the T24 cell suspension was added to the top portion of each well with a final cannflavin A concentration of 2.5 μM. Seven hundred microliters of McCoy’s 5A medium containing 10% FBS was added to the bottom portion of each well. After 24 h, media and cells that did not invade were removed from the inside of the insert with a dampened cotton swab. Wells were placed in methanol for 10 min and then transferred into a 3.5 g/L Crystal Violet in 2% ethanol solution for 10 min. Wells were then rinsed with dH₂0 and left to dry overnight. Cells that invaded through the Matrigel were counted using an Olympus CKX41 light microscope. Percent invasion was calculated by dividing the number of cells invaded in each condition by the number of cells that migrated in the control.

Mice were culled by CO₂ asphyxiation and the innervating whole DRG were removed as soon as possible and fixed in 2 % paraformaldehyde/0.5 % glutaraldehyde for 1 h on ice, permeabilised and stained with 1 mg ml⁻¹ X-gal for the detection of β-gal expression as previously described [27 (link)]. The DRG were then visualised and photographed using an Olympus CKX41 light microscope and Olympus DP20 camera. The number of β-gal⁺ cells was determined with the aid of ImageJ software [55 ].

Kidney sections were boiled in citric acid buffer (10 mM, pH 6.0) for 20 min for antigen retrieval. The sections were blocked in 10% normal horse serum and incubated with a primary antibody (1:100 diluted) against Wilms’ Tumor Protein 1 (WT1) from Boster Biological Technology (Pleasanton, CA, USA) overnight at 4 °C. The sections were incubated with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were incubated in avidin-biotin-peroxidase complex solution (ABC solution; Vector Laboratories) for 30 min and then developed using a 3,3′-diaminobenzidine (DAB) Peroxidase Substrate Kit (Vector Laboratories) for 30 s. The sections counterstained with hematoxylin were analyzed by using a CKX41 light microscope (Olympus). The number of stained nuclei was counted from 10 images of 200× microscopic fields per kidney section from each group.