Blocking solution

To determine the cellular localization of Cyp17a2 in the testis and head-kidney of tilapia, the testes and head-kidneys from wild-type males at different development stages were dissected and fixed in Bouin’s solution for 12 hours at room temperature, then dehydrated and embedded in paraffin. The tissue blocks were then sliced into 5-μm-thickness sections, then the sections were treated in a blocking solution (5% BSA diluted in 1x PBS) (Sangon Biotech, China), incubated with the primary antibody against Cyp17a2 overnight at 4°C and rinsed with 1x PBS five times for 5 min per wash. In this study, the anti-Cyp17a2 polyclonal antibody was diluted at 1:1000 before use. As a negative control, the primary antibody was replaced with normal rabbit serum. The slides were then incubated with anti-rabbit immunoglobulin G (diluted at 1:1000) at room temperature for 1 h, and then rinsed with 1x PBS three times for 5 min per wash. Immunoreactive signals were visualized using diaminobenzidine tetrachloride (DAB) (Sigma, Germany) as the substrate. Sections were then counterstained with hematoxylin. Finally, all the images for these sections were acquired with an Olympus BX5 light microscope (Olympus, Japan).

PC paraffin tissue sections (Shanghai Outdo Biotech (HPanA020PG02)) were dewaxed and rehydrated using xylene and gradient alcohol, respectively, and rinsed twice with PBS-T for 5 min each. Endogenous peroxidase was removed with 3% H₂O₂ (in methanol), and antigen repair was performed using EDTA solution (0.05 M, pH 8.0) at 85° C. Next, the tissue sections were cooled to room temperature, rinsed thrice with PBST for 5 min each, and blocked for 2 h with 3 mL of blocking solution (Sangon Biotech, China). The specimen was incubated with the decorin primary antibody (Abcam, Cambridge, MA, USA), followed by the HRP-labeled secondary antibody. The tissue sections were visualized using the DAB color development kit (Solarbio, Beijing, China), and counterstained with hematoxylin and eosin for 2 min, fractionated with HCl for 30 s, blued with tap water, and finally sealed with glycerin gelatin sealing tablets (Solarbio). Nikon E80i microscope (Nikon, Tokyo, Japan) was used to observe and photograph the sections and Image J was used to quantify the signal of decorin for each individual stage (I-III).

All the BMDC samples were lysed in RIPA buffer (Beyotime) on ice for 30 min, and equal amounts of protein lysates were subjected to SDS-PAGE. The proteins were then transferred to nitrocellulose (NC) membranes, which were blocked in blocking solution (Sangon Biotech). After washing, each NC membrane was incubated with the corresponding primary antibody (phospho-JNK, JNK, phospho-p38, p38, p65, IκBα, LaminB1, p-ERK and ERK) overnight at 4°C. Following incubation with the secondary antibody linked to HRP, the bands were visualized with an ECL detection system as per the manufacturer's instructions.