Ta 250 pm1x

Tissue sections (4 μm) were cut from formalin-fixed, paraffin-embedded blocks. These sections were baked onto poly-lysine slides at 60°C before one set was stained with H&E to investigate morphology and check suitability for further investigation. The following antibodies were used for IHC: Ki67 (RM-9106; Thermo Fisher Scientific) and ASS1 (cat. no. 70720; Cell Signaling). The sections were deparaffinized in xylene, rehydrated through graded ethanols, and washed in deionized water before undergoing heat-induced epitope retrieval using a Dako pretreatment module. All sections were heated for 25 min at 98°C in 10 mM sodium citrate (pH 6) retrieval buffer (TA-250-PM1X, Thermo Fisher Scientific). Sections were washed in TBS with Tween (TBST). The staining took place on a Dako Autostainer Link 48. The antibodies against Ki67 and ASS1 were used at 1:100 and 1:1000 dilutions, respectively. Sections were then incubated with Dako EnVision rabbit secondary antibody, washed with TBST, and stained with Dako liquid diaminobenzidine. The sections were counterstained with hematoxylin, taken through graded alcohols, xylene, and then a glass coverslip applied with DPX mountant for microscopy (CellPath). Slides were scanned with a Leica SCN400F slide scanner (Leica Biosystems, Wetzlar, Germany), and images were acquired through the Leica Digital Image Hub.