Protein Extraction from FFPE Tissues

Protein extraction from formalin-fixed paraffin-embedded tissues was performed according to Guo and co-workers [47 (link)]. Briefly, paraffin blocks were cut (approximately 10 µm), deparaffined in xylene and rehydrated in decrescent graded alcohol (100, 90, 70, 50, 30% v/v). Samples were resuspended in 20 mM Tris pH 9 and 2% SDS and boiled for 20 min at 100 °C and 2 h at 80 °C. After centrifugation, supernatants were collected, and protein content quantified by Quick Start Bradford (Bio-Rad, Hercules, CA, USA). Fifty μg of proteins from each sample were diluted in Laemmli buffer and boiled for 5 min. Samples were subsequently resolved on SDS-PAGE and transferred to PVDF membranes. Membranes were blocked for 1 h at RT with 5% non-fat dry milk in TBS buffer (20 mM Tris and 105 mM NaCl) containing 0.1% Tween-20 (block solution) and then incubated overnight with primary antibody dissolved in block solution. The day after, membranes were washed three times in TBS containing 0.1% Tween-20 (TBST) and incubated in secondary anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA, USA) diluted 1:10,000 in block solution. Immunostained bands were detected by a chemiluminescent method (Clarity Western ECL Substrate, #1705061, Bio--ad, Hercules, CA, USA). Primary antibodies (α-SMA and tubulin) were diluted 1:1000 in block solution.