PKM2 Immunohistochemistry Staining Protocol

IHC was used to detect PKM2 staining as previously described [13 (link)]. In brief, the sections were stained with an anti-PKM2 antibody (Cell Signalling Technology, 1:1000) and incubated overnight at 4°C. The sections were then processed using a MaxVision™ HRP-Polymer Anti-Rabbit IHC Kit (Maixin, Fuzhou, China), developed with a DAB Horseradish Peroxidase Colour Development Kit (Maixin, Fuzhou, China) and counterstained with haematoxylin. The degree of immunostaining was scored according to both the proportion of positively stained tumour cells (0∼3) and the staining intensity (0∼3), as previously described [29 (link)]. The staining index was calculated by multiplying the staining intensity score by the proportion of positive tumour cells, and the results were 0, 1, 2, 3, 4, 6, and 9. An optimal cut-off value (median) was identified as follows: a staining index score of >4 was used to define high PKM2 expression, and a score ≤4 was defined as low PKM2 expression.