Protocol for 3D tumor spheroid generation, immunostaining, and imaging through comparative approaches.

3D tumor spheroids are complex systems that can simulate protein localization in vivo. Here, we present a protocol for spheroid generation coupled with immunolabeling strategies. We describe steps for spheroid formation, block preparation, and immunostaining. We then detail procedures for image capturing and analysis. We provide guidance in maintaining spheroid cytoarchitecture, ensuring optimal antigen retrieval, and achieving consistent antibody accessibility. This protocol enables researchers to choose the most suitable approach for high-quality imaging to study 3D protein localization. For complete details on the use and execution of this protocol, please refer to Adhikari et al.(1).