Multiplex Immunohistochemistry Tissue Staining

Multiplex staining of primary tissues was performed by using Opal^TM 7-color multiplex IHC kit (Akoya Biosciences, NEL861001KT) according to the manufacturer’s instruction^{65 (link)}. Briefly, the slides were baked for approximately 1 h at 68 °C followed by de-paraffinization and rehydration. For each staining cycle, the slides were treated with the retrieval of antigen, blocking, and primary antibodies incubation, followed by HRP-conjugated secondary antibody incubation and Opal tyramide signal generation (Detailed information of antibodies and corresponding Opal fluorophores were provided in Supplementary Table S1). Then the slides were stripped with a retrieval solution as required before the next round of staining. The above process was repeated until all markers were completed. Once all markers were labeled, slides were counterstained with DAPI (Akoya Biosciences) and scanned using the PerkinElmer Vectra3® Polaris^TM platform. The multispectral images obtained were unmixed using the inForm Advanced Image Analysis software (inForm 2.4.1; Akoya Biosciences, USA).