Ds fi1

Next, because hepatic lipid accumulation leads to insulin impairment and insulin insensitivity, we assessed how HFHC diet consumption and solTNF signaling impact ectopic lipid deposition in liver tissue [28 (link)] Liver tissue from the left lobe was fixed in 4% paraformaldehyde/PBS and cryoprotected in 30% sucrose solution. Tissue was frozen in OCT, sectioned (10 μm), and stained with Oil Red O (150678, Abcam) according to the manufacturer’s instructions. Images were obtained using a Nikon Eclipse 90i microscope with a DS-Fi1 (Nikon) camera and Nikon NIS-Elements AR 3.10 software, magnification × 40.

After the mice were euthanized, the livers were collected and stored in 10% formalin. The liver tissue was randomly chosen for sections processing. Microscopic slides were obtained after preparation of wax blocks and staining with hematoxylin and eosin (Leica, IL, United States). Tissue sections were observed under a microscope for differential cell counts. The histopathological sections from each mouse were analyzed quantitatively for the different cell populations percentage, including Kupffer cells, apoptotic cells, mononucleated hepatocytes, and binucleated hepatocytes. The sections were observed under a Nikon Eclipse 50i light microscope, and the image was captured with a Nikon DS-FI1 digital camera. Each slide contained three sections obtained at 15-μM intervals, and random six different areas were examined with 3 × 10⁴ μm overlaid grid. The cells were scored with NIS elements D image analysis software (Nikon, New York, NY, United States). Each (mouse) slide was examined with six different areas of 18 × 10⁴ μm² and each group examined for 54 × 10⁴ μm². The hemorrhagic areas were observed in 20 magnifications (to include a wider area of 216 × 10⁴ μm² per group). All the mean values were analyzed using one-way ANOVA, IBM SPSS statistics version 23, and represented in bar chart.

We used an inverted polarising microscope (Ti-U, Nikon) with ×100 oil immersion objective (NA = 1.4), Nikon DS-Fi1 digital camera (pixel size 3.4 × 3.4 μm). Kossel diagrams were recorded in conoscopic observation mode from a single domain with fully open illumination aperture diaphragm and fully closed eld diaphragm. Interference filters at 514.5 nm with 5 nm transmission bandwidth were used for wavelength selection. Due to low intensity of the reflected light and narrow filter bandwidth exposure times were from 4 to 30 seconds depending on wavelength and sample region.

The aim of this experiment was to monitor pupal development after 20E-treatment. The pupae samples used for this experiment were collected from the wild at the end of November, 2014, thus, they had already entered into pupal diapause. 20E was applied as reported above to break the pupal diapause. Following this treatment, pupae from all groups were dissected to record their developmental process. This was done by taking photomicrographs (Nikon DS-Fi1) every 3 days under a dissecting microscope (JSZ5BS). The pupal development was continuously monitored through this process until the adult emerged.

Individuals used for measurements were stored in 70% ethanol at 4°C until photographed on millimetre paper using a Nikon SMZ800 stereo microscope with Nikon DS Fi1 camera attachment. Measurements on these photographed specimens were made with ImageJ (v. 1.53 k, see electronic supplementary material, figure S2 for definition of measurements). All statistical analysis was performed in R (v. 4.0.3). ANOVAs, t-tests, Pearson's χ² and generalized linear models were performed within the R set of core functions (stats 4.0.3). All code and data are available in the electronic supplementary material. The estimation of pairwise sequence identity of the inr genes coding sequences was done with Muscle (v. 3.8.425 (alignment in Geneious (v. 2021.1.1) using standard parameters.