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The cellular localization of Lv-AE3 mRNA was performed in the gill with high transcript levels by ISH as described previously (Ruan et al., 2020 (link)). The digoxigenin (DIG)-dUTP-labeled DNA probe targeting Lv-AE3 was generated by using a DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Switzerland). The slices incubated by DIG-labeled probe were incubated with horseradish peroxidase (HRP)-conjugated anti-DIG antibody (Roche), and the ISH signal was developed by a diaminobenzidine (DAB, MXB Biotechnologies) reaction and the nucleus were stained with hematoxylin. The Lv-AE3 mRNA expressed cells in the gill were observed and recorded with a Leica DM-IRB light microscope (Leica, Germany).

Paraffin-embedded sections of the CCA were cut for each group of rats, routinely dewaxed, hydrated, and heated in citrate antigen retrieval solution for antigen retrieval. The sections were blocked with 5% bovine serum albumin (BSA) and then incubated with an antiproliferating cell nuclear antigen (PCNA) polyclonal antibody (1 : 200; Proteintech, China) and anti-SMα-actin polyclonal antibody (1 : 200; CST, USA) overnight at 4°C followed by biotin-labeled goat anti-rabbit IgG (1 : 2000; Abcam, USA) at 37°C in the dark for 30 min. Then, diaminobenzidine (DAB; MXB, China) was used for color development, images were captured and saved with the ISCapture system, and Image-Pro Plus 6 image processing software was used for data analysis.