In modern life science research, the complexity of tissue microenvironments poses higher demands on scientific research. Multiplex immunofluorescence (mIHC) technology, with its advantages of simultaneously detecting multiple targets and preserving spatial information, has become an indispensable tool in fields such as tumor immunology, neuroscience, and stem cell research.
Over the past few decades, immunohistochemistry (IHC) has been the standard tool for tumor tissue research: from HER2, Ki-67 to PD-L1, it has allowed researchers and pathologists to clearly see the distribution of individual proteins in tissues. However, as tumor research enters the "spatial biology era," information from single markers is far from sufficient.
This study combined sequential stepwise staining with tyramide signal amplification detection technology, successfully establishing a multiplex immunofluorescence staining system suitable for fully automated staining platforms, breaking through the technical barrier that traditional automated staining cannot use same-species primary antibodies for multi-target detection.
In recent years, Multiplex Immunohistochemistry (mIHC), with its ability to simultaneously detect multiple markers and analyze spatial information, has become an important tool for tumor microenvironment research, immunotherapy evaluation, and spatial pathology studies. However, compared to traditional IHC, the mIHC experimental workflow is more complex. From antibody screening to Panel design, to TSA staining and image analysis, every step can affect the final result.
With the rapid development of tumor microenvironment research, spatial biology, and precision pathological diagnosis, Multiplex Immunofluorescence (mIHC) has become an important tool for tissue sample analysis. Compared with traditional immunohistochemistry (IHC) and conventional immunofluorescence (IF), mIHC enables simultaneous detection of multiple protein markers on the same tissue section, allowing cell phenotype identification, spatial localization analysis, and cell-cell interaction studies.
TSA (Tyramide Signal Amplification) kits are high-sensitivity immunoassay systems developed based on the principle of HRP enzyme-catalyzed fluorescent tyramide molecule deposition. Its core mechanism is to use HRP-labeled secondary antibody to catalyze the activation of fluorescent tyramide, causing it to undergo covalent deposition around the target antigen, thereby achieving local high-density fluorescence enrichment and significantly improving detection sensitivity.
The iterative advancement of spatial pathology technology has completely broken through the limitations of traditional pathological detection, including single markers, weak signals, and missing spatial information, becoming a core supporting technology for tumor microenvironment analysis, disease mechanism research, and precise pathological diagnosis.
This study aimed to systematically analyze the immune microenvironment of lung cancer brain metastasis under different genetic backgrounds and TKI treatment states using single-cell transcriptome sequencing and multiplex immunohistochemistry, confirm CTLA4 as a key target to overcome TKI resistance, and demonstrate in mouse models of EGFR-sensitive and resistant mutant lung cancer brain metastasis that sequential use of EGFR-TKI and CTLA4 blockade is significantly more effective than TKI monotherapy or TKI combined with PD-1 blockade, providing a novel therapeutic strategy for overcoming TKI resistance in lung cancer brain metastasis.
