2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi

The hemocytes were collected and resuspended in an L15 medium. The suspension was deposited on clean slides (a drop on each) in the wet chamber. After the monolayer was sedimented on the side, the hemocytes were fixed with 4% paraformaldehyde (PFA) for 10 min. After being washed with L15 medium for 5 min, the hemocytes were blocked by incubating in 3% BSA at 37°C for 30 min. The supernatant was then removed, and the dishes were incubated with 500 µl of anti-LC3 (diluted 1:200 in 3% BSA) at 4°C for 2 h. After washing three times with L15 medium, the hemocytes were incubated with Alexa Fluor 488-labeled goat anti-Rabbit secondary antibody (diluted 1:200 in 3% BSA) at 37°C for 1 h. After washing another three times with an L15 medium, the hemocytes were incubated with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Solarbio Life Sciences, China, diluted 1:1,000 in 3% BSA) to stain the nucleus. The slides were mounted on buffered glycerin (50%) and observed using a fluorescence microscope (ZEISS, Germany) after the final three times of washing with the L15 medium. ImageJ was used to calculate the fluorescence signals of CgLC3 in the hemocytes.

Cell type and distribution were determined by H&E staining and immunohistochemical staining. The specific experimental steps refer to the previous literature of our team (28 (link)). Primary and secondary antibodies are listed in Table S2. For antigen retrieval, the paraffin tissue sections were immersed in citrate buffer (pH 6.0), and placed in a pressure cooker until steam was generated for 3 min. To block non-specific peroxidase, a 3% hydrogen peroxide and methanol solution was applied to tissue sections for 10 min, following incubation in goat serum (Solarbio Life Science, Beijing, China, S9070) for 30 min. Tissue sections were then incubated with diluted primary antibody (Table S2) at 4°C overnight and washed twice with PBS. Fluorescent dye-labeled or HRP-labeled secondary antibodies were applied to the tissue sections for 30 min, followed by three PBS washes. The nuclei were counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Solarbio Life Science, Beijing, China, C0065) or DAB (Dako, Glostrup, Denmark, 20052898) and hematoxylin (Scientific Phygene, Fuzhou, China, PH1464). Images of all sections were captured using a Leica DM2500 microscope.

RAW 264.7 cells were inoculated to 6 well cell culture plates, and the next day the medium was replaced with material leach liquors containing LPS (1 µg mL⁻¹) for 24 h. The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized by 0.5% Triton X‐100 for 15 min. After three washes with PBS, cells were blocked using 10% normal goat serum. Subsequently, cells were incubated with anti‐CD206 (host: rabbit) and anti‐iNOS (host: mouse) primary antibodies (1:500; Abcam, Cambridge, UK) at 4 °C overnight, washed with PBS and incubated with Alexa Fluor 594‐conjugated goat anti‐rabbit IgG secondary antibody and Alexa Fluor 488‐conjugated goat anti‐mouse IgG secondary antibody (1:500; Proteintech, Wuhan, China) in the dark for 1 h at 37 °C. Nuclei were redyed using 2‐(4‐Amidinophenyl)−6‐indolecarba midine dihydrochloride (DAPI; Solarbio). Images were observed under a fluorescence microscope (Leica DMi8, Wetzlar, Germany) in the darkroom.