For researchers in biology, medicine, and pharmaceutical science, three core immunological detection techniques are virtually unavoidable: ELISA, Western Blot (WB), and Immunofluorescence (IF). These three techniques, based on the core principle of antigen-antibody specific binding, are fundamental methods for protein expression detection, sample qualitative and quantitative analysis, and molecular localization analysis, spanning the entire workflow of cell experiments, tissue detection, drug screening, and clinical sample validation.

This article will guide you through the core steps of an mIHC project from raw sections to top-tier journal figures, helping you thoroughly understand the research logic and application value of mIHC.

To truly understand tumors and optimize tumor research and treatment, the first step is to recognize the core cellular components of the tumor microenvironment. This is precisely why multiplex immunohistochemistry (mIHC) technology has become the core tool for tumor research.

This study systematically confirmed through multiplex immunofluorescence single-cell imaging and multidimensional statistical analysis that multi-marker combined T cell profiling has significant clinical advantages compared to traditional single-marker detection, successfully solving the challenge of prognostic stratification for stage III colorectal cancer patients undergoing FOLFOX chemotherapy.

Published in *Advanced Science*, this research was completed by the teams of Min Zhou and Danni Zhong from the First Affiliated Hospital, Zhejiang University School of Medicine. They constructed a colon-targeted CV@PA-gel synergistic platform and clarified its mechanism for intervening IBD gut-brain comorbidity: CV@PA-gel treatment, a novel, safe and effective intervention, can maintain intestinal barrier integrity and reduce gut microbiota translocation, further block microglia-induced A1 astrocyte transformation, and ultimately alleviate DSS-induced depressive-like behaviors and cognitive dysfunction.

Traditional IHC relies on chromogenic substrates for qualitative detection, while conventional IF depends on direct fluorescent labeling. Although seemingly mature, these methods have insurmountable technical bottlenecks in detecting low-abundance targets, co-localizing multiple markers, and adapting to diverse sample types.

This article systematically elaborates the specific application scenarios and clinical value of multiplex immunostaining technology in tumor immune microenvironment research, combined with multiple research examples.

This study introduces tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) technology, aiming to break through the limitations of traditional detection techniques and establish a standardized technical system that can achieve simultaneous detection of multiple markers, precise immune cell classification, and spatial atlas analysis on a single paraffin-embedded tumor tissue section, providing reliable technical support for in-depth research on the tumor immune microenvironment, immune biomarker discovery, and the implementation of precision immunotherapy.

Redox homeostasis denotes the maintenance of the balance between oxidants and antioxidants. Although excessive oxidant production causes oxidative damage to cellular biomolecules and results in oxidative distress, lack of adequate oxidant levels impairs crucial signalling processes and results in reductive distress.