Dab horseradish peroxidase colour development kit

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.

Immunohistochemistry (IHC) was used to detect HK2, as previously described [12 (link)]. Briefly, paraffin sections were deparaffinized using xylene and rehydrated using a series of alcohol solutions. Antigen retrieval was performed by treating the sections with boiling citrate buffer (pH 6.0) for 4 min in a pressure cooker. Endogenous peroxidase activity was blocked by treatment with 3% H₂O₂ for 15 min. The sections were then incubated with an anti-HK2 antibody (Atlas, 1:250) overnight at 4°C. The sections were incubated with the secondary antibody using a MaxVision™ HRP-Polymer anti-Rabbit IHC Kit (Maixin, Fuzhou, China); colour was developed using a DAB Horseradish Peroxidase Colour Development Kit (Maixin, Fuzhou, China), and the sections were counterstained with haematoxylin. The degree of immunostaining was scored according to both the proportion of positively stained tumour cells and the staining intensity, as described previously [30 (link)]. We evaluated HK2 expression by determining the staining index (staining intensity score × proportion of positive tumour cells), which resulted in scores of 0, 1, 2, 3, 4, 6 and 9. An optimal cut-off value (median) was identified, with a staining index score of >4 defining a high level of HK2 expression and a staining index score of ≤4 defining a low level of HK2 expression in the tumours.