4 6 diamidino 2 phenylindole dapi

Wheat seeds were removed from the spike then treated with 4% paraformaldehyde at 4°C overnight and gradient-dehydrated. The prepared grain tissues were then embedded in paraffin, cut into 7-µm sections, adhered to gelatin-coated glass slides, and dried at 37°C overnight. The slides were then dewaxed, gradient-dehydrated, and digested with 20 µM proteinase K at 37°C for 10 min before blocking in 2% BSA at 37°C for 30 min. Rabbit anti-ATG8 antibody was then added before incubating the slides at 47°C overnight. They were then washed three times with PBS before adding 1 µL secondary antibody (goat anti-rabbit-Alexa Fluor 555 antibody in 10 ml blocking buffer), and incubating at 37°C for 1 h. The nuclei were then stained with 4′, 6-diamidino-2-phenylindole (DAPI) (AnaSpec Inc., San Jose, CA, United States) at room temperature for 10 min. Fluorescence was observed with a fluorescence microscope (HT7700, Hitachi, Tokyo, Japan).

An immunocytochemical assay was performed to visualize the chitinous materials in the shrimp cuticle. The chitin-binding domain (CBD) of Bacillus circulans WL-12 Chitinase A1 was recombinantly expressed in bacterial cells using the pET32a (+) vector, purified using affinity chromatography (see below), and used as a probe to bind to chitinous materials. This probe can be visualized by using an anti-His tag antibody and fluorescein isothiocyanate (FITC)-conjugated secondary antibody. The cryosections (6 μm) were treated with the probe solution (0.1 mg/mL) for 12 h. Afterwards, mouse anti-His tag monoclonal antibodies (Abbkine, Wuhan, China; ABT2050; 1:100 dilution) were used to target the recombinant CBD, which was fused with a 6His tag. After washing with PBS, FITC-conjugated goat anti-mouse (Abbkine; A22110; 1:1000 dilution) was added and incubated for 2 h in the dark. Then, 4′,6-diamidino-2-phenylindole (DAPI, AnaSpec, Fremont, CA, USA; AS-83210; 1:1000 dilution) was used to stain the nuclei in the epidermis. Finally, the slides were washed with PBS and observed under a Zeiss LSM 900 confocal microscope (Carl Zeiss, Jena, Germany). The images were analyzed and presented using ZEN software (Zeiss).

B16F10 (3 × 10⁴) cells, seeded onto a poly l-Lysin-coated 18-mm slide glass, were treated with 10 µM GIF-2209 for 24–48 h. Cells were fixed with 4% paraformaldehyde and 0.025% Triton-X 100 (Nacalai Tesque, Kyoto, Japan). After blocking, cells were incubated with the primary antibody (anti-TYR antibody as described in [8 (link)], anti-TYRP-1 antibody was from Santa Cruz Biotechnology, Dallas, TX, USA, and anti-CD63 antibody was from MBL) (1/5000), then the secondary antibody (goat anti-rabbit IgG H&L [Alexa Fluor^® 488] and goat anti-mouse IgG H&L [Alexa Fluor^® 594], Abcam, Pleasanton, CA, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, ANASPEC INC., CA, USA). Finally, cells were placed on a glass plate. Cell Navigator^® Lysosome Staining Kit Red Fluorescence was acquired from ATT Bioquest (Sunnyvale, CA, USA), and the cells were stained according to the manufacturer’s protocol.