Chemmate envision hrp labelled polymer system

Formalin-fixed, paraffin-embedded tumor sections (2 μm) were de-paraffinized in xylene and rehydrated through a 100-95% ethanol gradient. Sections were stained with Hematoxylin and Eosin followed by a series of dehydration steps via ethanol washes, cleared with xylene and mounted using Safemount (Bio Optica, Milan, Italy). Heat induced epitope retrieval was done with EDTA buffer pH 8.0 (1 h, 98 °C) and 0.3% H2O2 was used to block endogenous peroxidases. Sections were then washed in TBS (pH 7.4) and incubated with the specific primary antibody diluted in TBS with 1% bovine serum albumin (BSA) (1 h, RT). Protein signal was revealed by ChemMATE EnVision HRP Labelled Polymer system (DAKO, Glostrup, Denmark) followed by diamino-benzydine as chromogen and Hematoxylin as counterstain. After re-peating a series of dehydration steps via ethanol washes, sections were cleared with xylene and mounted using Safemount (Bio Optica, Milan, Italy). Images were acquired by an inverted light microscope (Olympus IX50; Olympus, Tokyo, Japan) using cellSens Software (Olympus, Tokyo, Japan).