Ab233434

Typical CRC tissues and normal colorectal mucosal tissues were selected. The paraffin-embedded tissues were cut into 4-μm thick sections, dewaxed using xylene, rehydrated through gradient ethanol, and then heated using a pressure-cooker for antigen retrieval. Antigenic repair was performed with citric acid or repair solution, and endogenous peroxidase blocked with 3% H₂O₂-methanol (prepared with 30% H₂O₂ 10 mL + methanol 90 mL). The sections were then incubated overnight at 4 °C with the following primary antibodies: Rabbit polyclonal anti-PGAM5 (1:200 dilution, ab126534; Abcam, United Kingdom) and rabbit polyclonal anti-Parkin (1:400 dilution, ab233434; Abcam, United Kingdom). Subsequently, sections were incubated with the secondary antibody for 40 min at 37 °C. Next, after washing in phosphate-buffered saline, the sections were stained with diaminobenzidine and then counter-stained with hematoxylin. The sections were then air-dried, dehydrated, and mounted. CRC tissues with intense immunoreactivity to PGAM5 and Parkin were used as positive controls; in the negative control, the primary antibody was replaced with phosphate-buffered saline.

Protein was extracted from the six matched pairs of colorectal adenocarcinoms and corresponding paracancerous tissues with the RIPA lysis buffer containing PMSF protease inhibitors and was quantified by using the BCA assay (Beyotime Institute of Biotechnology, China). Samples of 50 μg of protein were loaded and resolved by SDS-PAGE followed by transfer to a PVDF membrane. Non-specific binding was blocked using 5% bovine serum albumin in Tris Buffered Saline with Tween 20 (TBS-T, pH 7.6) for 2 h. Subsequently, incubation with primary antibodies, rabbit polyclonal anti-PGAM5 (1:1000 dilution, ab126534; Abcam, United Kingdom), rabbit polyclonal anti-Parkin (1:1000 dilution, ab233434; Abcam, United Kingdom), and glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase, 1:1000 dilution, FL-335; Santa Cruz Biotechnology, CA, United States) was undertaken overnight at 4 °C. After extensive washing, the membranes were incubated with anti-rabbit IgG secondary antibody (1:5000) and the immunoreactive bands were detected with ECL-Plus chemiluminescent detection HRP reagents (Beyotime Institute of Biotechnology, China) using the Microchemi 4.2 Bio-imaging system. The Western blots were quantified by densitometric analysis using ImageJ software (Version 1.46r). The experiments were repeated three times under the same experimental conditions.