Re7150 k

Tumor tissue sections were deparaffinized in xylene and rehydrated in a series of graded ethanol. Histological analysis of tumor tissues was carried out using hematoxylin and eosin (H&E) staining (HAE-1, ScyTek Laboratories, Logan, Utah, USA). Immunohistochemical staining was performed using a Novolink polymer detection system (RE7150-K, Leica Biosystems Newcastle Ltd., Newcastle, UK) according to the manufacturer's instructions. Heat-induced antigen retrieval was performed in Tris–EDTA buffer (pH 9.0) containing 10 mM Tris-base, 1 mM EDTA solution and 0.05% Tween-20 for 30 min. Endogenous peroxidase was quenched with peroxidase block, and non-specific protein binding was blocked using protein block. Subsequently, the slides were incubated with the appropriate primary antibodies overnight at 4 ℃, followed by incubation with the provided Novolink polymer for 1 h. The primary antibodies were diluted in protein block and the dilutions were as follows: anti-BiP (1:100), anti-VEGF (1:100), and anti-CD31 (1:100). The signals were developed with 3,3′-Diaminobenzidine (DAB) solution, and the nuclei were counterstained with hematoxylin. Quantification of histochemical staining was performed using ImageJ software.

The IHC staining was performed on 4 μm tissue microarrays sections using a Novolink polymer detection system (RE7150-K, Leica Biosystems, Newcastle, UK), as previously described [12 (link)]. Evaluation of membranous staining for SLC7A5 1:200 (EPR17573, Abcam, Cambridge, UK) and SLC3A2 1:2000 (HPA017980, Sigma-Aldrich, Dorset, UK) were based on a semi-quantitative assessment of invasive tumour cells using a modified H-score as previously described [11 (link),12 (link)]. TMA cores were only assessed if the tumour burden was >15%.