The M-HFn nanoparticle-based IHC assay for GC tissues was performed as previously reported12 (link). As negative controls, the sections were processed using the same protocol, except that they were not incubated with the M-HFn nanoparticles. The staining of TfR1 was examined and scored by two independent pathologists under microscopy, who were blind to the patient clinical data. Immunoreactivity score together with the staining intensity were used to assess the staining of TfR1. Finally, the staining levels of TfR1 expression were ascribed to − and +, low expression; ++ and +++, high expression.
For IHC, primary antibodies were used to detect anti-transferrin receptor antibody (anti-TfR1, Abcam, Cambridge, UK), PCNA (Dako, Carpinteria, CA, USA), and Ki67 antibody (Abcam, Cambridge, UK). For IF, primary antibodies were used to detect CD44 (Dako, Carpinteria, CA, USA) and 1B50-1 (anti-CACNA2D1), gifted by Prof. Zhang, Peking University, with visualization using fluorescein AF488 and AF594 (Jackson Alexa Fluor). DAPI was used to detect nuclei.
For IHC, primary antibodies were used to detect anti-transferrin receptor antibody (anti-TfR1, Abcam, Cambridge, UK), PCNA (Dako, Carpinteria, CA, USA), and Ki67 antibody (Abcam, Cambridge, UK). For IF, primary antibodies were used to detect CD44 (Dako, Carpinteria, CA, USA) and 1B50-1 (anti-CACNA2D1), gifted by Prof. Zhang, Peking University, with visualization using fluorescein AF488 and AF594 (Jackson Alexa Fluor). DAPI was used to detect nuclei.