Antigen retrieval pt module

For immunohistochemistry (IHC), 4-µm sections were dewaxed, hydrated and heat-treated in 1 mM EDTA pH 8 in an antigen retrieval PT module (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 95 °C for 20 min. Sections were then stained with the following antibodies: Rabbit monoclonal antibody to vimentin (Clone SP20) 1:200 dilution (Spring Bioscience Corp., Pleasanton, CA, USA); goat polyclonal antibody to Snail (ab53519), 1:200 dilution (Abcam, Cambridge, MA, USA); mouse monoclonal antibody against TGF-β2 (220ct16.4.3.1), 1:40 dilution (Cell Marque Corp., Rocklin, CA, USA). The staining procedure was performed according to the manufacturers’ protocols using an Autostainer 480 (Thermo Fisher Scientific Ltd., Vantaa, Finland). The visualization system included UltraVision LP Detection System HRP Polymer & DAB Plus Chromogen (Thermo Fisher Scientific Inc., Waltham, MA, USA) and hematoxylin counterstaining. For the negative control sections, primary antibodies were replaced with phosphate-buffered saline and processed in the same manner. Stained slides were evaluated by two pathologists (MS and OT). The number of Snail and TGF-β2 positive cells was counted in 10 randomly selected fields by light microscopy at 400× magnification. The percentage of positive cells was expressed relative to the total number of tumor or epithelial cells counted.

Decalcified and paraffin-embedded sections were dewaxed, hydrated, and heat-treated in 1 mM EDTA pH 8 in an antigen retrieval PT module (Thermo Fisher Scientific Inc., Waltham, MA) at 95°C for 20 min. Sections were incubated for 16 h at 4°C with the prediluted polyclonal antibody against Musashi-1 (Sigma-Aldrich, Barcelona, Spain) at 1 : 100 dilution to identify cellular expression. An automatic immunostainer (Autostainer 480, Thermo Fisher Scientific Inc.) was used for the immunohistochemical study, applying the peroxidase conjugated micropolymer method and developing with diaminobenzidine (Ultravision Quanto, Master Diagnóstica, Granada, Spain). Expression was assessed semiquantitatively on a scale of 0 to 3 (0: absence, 1: mild [<10% positive cells], 2: moderate [10 to 25%], 3: intense [>25%] in bone tissue, hyaline cartilage, joint capsule, synovium, ligaments, striated muscle cells, endothelial cells, and adipocytes). The variables were subsequently categorized in two groups (presence/absence of osteoarticular lesion), calculating the total Msi1 expression score for each group.