Ion sputter

We modified the sample preparation for SEM for the examination of VALT. Briefly, the mice were perfused with 2.5% GTA through the ventricle. The excised kidney was sliced and fixed with GTA and PFA for 4 and 12 h, respectively, to prepare the paraffin block. The paraffin blocks were cut at 5 mm thickness and mounted on a glass slide. The sections were deparaffinized, fixed, and post-fixed with GTA and osmium tetroxide. The specimen was coated with ion sputter (Hitachi; Tokyo, Japan) for 1 min and examined with an S-4100 SEM (SU 8000, Hitachi) with an accelerated voltage of 10 kV.

First, 0.6 g of gelatin (Meilunbio, Dalian, China) and 0.3 g of sodium alginate (Solarbio, Beijing, China) were weighed and added into 10 mL of sterile calcium-free PBS respectively, and fully dissolved at 70 °C. The gelatin-sodium alginate hydrogel was prepared by mixing the above solutions in a 1:1 (V:V) ratio. The final concentrations of gelatin and sodium alginate were 3% (w/v) and 1.5% (w/v). The gel solution was placed in a mold and in a 4 °C refrigerator. After molding, the gelatin-sodium alginate hydrogel was cross-linked by a certain amount of sterilized (2% w/v) CaCl₂ solution (Aobox, Beijing, China). After this, CaCl₂ was removed, and Gel-Alg hydrogel was washed in sterile PBS.
The hydrogel was freeze-dried in the freeze-dryer (Songyuan, Beijing, China) for 24 h, and then cut into small pieces. The surface of the sample was gilded by an ion sputter (Hitachi, Tokyo, Japan). The micromorphology of the freeze-dried gelatin-alginate hydrogel was observed by scanning electron microscopy (SEM) (Hitachi) under a 15 kV accelerating voltage.

Fourth-instar Wt^LW nymphs were microinjected with dsUbx, dsZfh1, or dsGfp, and allowed to molting into adults. After removing wings, female adults were placed on a stub and dried in an ion sputter (Hitachi) under vacuum. After gold sputtering, the samples were observed under SEM (TM-1000, Hitachi).

Three eggs from each group were randomly selected. The ultrastructure of an eggshell (0.5 ~ 1 cm²) was assessed using a scanning electron microscope. To facilitate membrane removal, eggshells were boiled in 2% NaOH for 10 min. Afterward, the shells were rinsed in water and dried for at least 24 h at room temperature. Thereafter, the eggshell samples were secured tightly on a conductive carbon film (double-sided adhesive) and analyzed using an ion sputter (Hitachi, Japan) for approximately 30 s. Subsequently, the eggshell cross-sections and inner and outer surfaces were observed using a scanning electron microscope (Hitachi, Japan). The effective thickness, mammillary thickness, and width of the mammillary knob were measured and averaged. The mammillary thickness was measured as the length from the top of the membrane to the bottom of the palisade. The average size of the mammillary knob was determined as follows: width = the length of the mammillary knob/the number of mammillary knobs. The percentages of effective thickness and mammillary thickness were calculated by measuring the ratio of the thickness of each layer to the total thickness. The cross-sectional images of each group were visualized under 200× magnification. For the inner and outer surface analysis, the images for each group were visualized under 100× magnification.

Surface morphologies of lyophilized RLX-SMEDDS, RLX powder, and mannitol were examined using scanning electron microscopy (SEM SU5000; Hitachi, Tokyo, Japan). The powdered samples were fixed on a brass stub with double-sided adhesive tape, coated with platinum under a vacuum using a Hitachi ion sputter, and imaged at an accelerating voltage of 20 kV.