Ab101500

Multiplex immunofluorescence staining with a four-color IHC kit (Absin, China) was performed on tumour samples from CBR group and NCB group patients for counting immune cells infiltrating different tumours. The slides were deparaffinized in xylene, rehydrated, and washed in ethylenediaminetetraacetic acid (EDTA)-sodium citrate buffer for microwave antigen retrieval. Endogenous peroxidase activity was blocked using an antibody diluent/blocking agent (PerkinElmer, USA). Primary antibodies CD3 (1:500, Abcam, ab699), CD8 (1:500, Abcam, ab101500), and CD68 (1:500, Cell Signalling Technology, E3O7V) were incubated at room temperature for 1 h. Polymer HRP Ms + Rb was incubated for 10 min at 37 °C. Subsequently, the slides were incubated at room temperature for 10 min with TSA fluorescent dyes (TSA520, TSA570, and TSA650) diluted in signal amplification solution. Antigen-antibody complexes were stripped by microwave treatment using 0.05% Tris–EDTA buffer. TSA single-stained slides were counterstained with DAPI for 5 min and then coverslipped. Three observers, blinded to the experimental design of each sample, evaluated random fields of view at 100x magnification and calculated the number of immune cells in each field. Images were acquired using a confocal laser-scanning microscope (Zeiss Microscopy, USA).