Three-micrometre sections were prepared from formalin-fixed, paraffin-embedded tissue samples. As described previously,19 (link) the sections were deparaffinised with xylene and rehydrated in four graded alcohol solutions (70%, 80%, 90% and 100%). Immunohistochemical (IHC) staining for GPC-1 was performed using rabbit polyclonal anti-GPC-1 antibody (Atlas Antibodies AB, Stockholm, Sweden, 1:400) and visualised with Envision ChemMate (Dako, Glostrup, Denmark) according to the manufacturer’s protocol.
Immunostaining was scored according to the intensity of the staining: 0, no staining; 1, normal staining; 2, strong staining. The ‘density’ of staining (termed the positivity score) was as follows: 1, indicates less than 50% positivity; 2, indicates more than 50% positivity. The final IHC score was determined by multiplying the intensity score by the positivity score, resulting in a maximum possible score of 4. These data were referred to as the GPC-1 score. Furthermore, we divided patients into two equally balanced groups by score.
Immunostaining was scored according to the intensity of the staining: 0, no staining; 1, normal staining; 2, strong staining. The ‘density’ of staining (termed the positivity score) was as follows: 1, indicates less than 50% positivity; 2, indicates more than 50% positivity. The final IHC score was determined by multiplying the intensity score by the positivity score, resulting in a maximum possible score of 4. These data were referred to as the GPC-1 score. Furthermore, we divided patients into two equally balanced groups by score.