Mayer s haematoxylin

Histological analysis was performed using the gonad of the individual sampled above in May 2022 for genome sequencing. Also, tissues of a male and a female collected in June 2022 were fixed in Bouin’s fixative and used for histological observations as a reference to the gonadal structure of each sex. All tissues were embedded in paraffin (Leica Biosystems, Nussloch, Germany), sectioned in 5 μm, and stained with Mayer’s haematoxylin (Fujifilm Wako Pure Chemical Co., Osaka, Japan) and eosin Y (Fujifilm Wako Pure Chemical Co.).

Dimethylnitrosamine (N-nitrosodimethylamine or DMN), 1,1,3,3-tetraethoxypropane (TEP), chloramine-T, p-dimethylaminobenzaldehyde, hydroxyproline, potassium chloride (KCl), and trichloroacetic acid (TCA) were purchased from Sigma (St. Louis, MO, United States); Thiobarbituric acid (TBA) was purchased from Lancaster Co. (Lancashire, United Kingdom). Hydrochloric acid and phosphoric acid were from Kanto Chemical Co., Inc. (Tokyo, Japan); n-butanol was purchased from J. T. Baker (Center Valley, PA, United States); Mayer's haematoxylin and isopropanol were obtained from Wako Pure Chemical Industries (Osaka, Japan); Goat anti-human connective tissue growth factor (CTGF) antibody, CTFG standard solution, rabbit anti-human CTGF antibody, and anti-rabbit immunoglobulin G horseradish peroxidase conjugate were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States), and DDB was purchased from Pharma King Co., Ltd. (Gyeonggi-do, Republic of Korea).