新的组织学技术需要用于检查人体组织中蛋白质的分布，这可以揭示细胞形态和疾病病理学之间的联系。空间蛋白组学通过确定免疫调节细胞和蛋白质的空间关系，并为新的癌症免疫治疗生物标志物的发现做出贡献，改变了肿瘤微环境的研究。然而，快速扩展的空间蛋白组学方法工具包尚未系统地应用于研究老年人脑部在健康和疾病状态下的病理变化。此外，由于固定程序和自发荧光，尸检的人脑组织存在着显著的技术问题，这限制了荧光方法的应用。本研究试图开发一种多重免疫组化方法（使用明场显微镜可视化免疫染色），即定量多重免疫组化与可视化显色增强区域蛋白定位（QUIVER），以解决这些技术挑战。使用QUIVER，利用色素去除和数字显微镜生成了一个十通道的伪荧光图像，以识别独特的分子小胶质细胞表型。接下来，本研究问：组织环境，特别是阿尔茨海默病的淀粉样斑块和神经纤维缠结是否对小胶质细胞的细胞和分子表型有任何影响。 QUIVER使用数字病理学工具可视化了五个分子小胶质细胞/巨噬细胞表型。可识别的反应性和稳态小胶质细胞/巨噬细胞表型表现出空间极化，分别远离和靠近淀粉样斑块。然而，小胶质细胞形态外观并不总是对应分子表型。这项研究不仅揭示了小胶质细胞的生物学，还提供了QUIVER，一种检查老年人脑部病理变化的新工具。©2023年。作者。

New histological techniques are needed to examine protein distribution in human tissues, which can reveal cell shape and disease pathology connections. Spatial proteomics has changed the study of tumor microenvironments by identifying spatial relationships of immunomodulatory cells and proteins and contributing to the discovery of new cancer immunotherapy biomarkers. However, the fast-expanding toolkit of spatial proteomic approaches has yet to be systematically applied to investigate pathological alterations in the aging human brain in health and disease states. Moreover, post-mortem human brain tissue presents distinct technical problems due to fixation procedures and autofluorescence, which limit fluorescence methodologies. This study sought to develop a multiplex immunohistochemistry approach (visualizing the immunostain with brightfield microscopy). Quantitative multiplex Immunohistochemistry with Visual colorimetric staining to Enhance Regional protein localization (QUIVER) was developed to address these technical challenges. Using QUIVER, a ten-channel pseudo-fluorescent image was generated using chromogen removal and digital microscopy to identify unique molecular microglia phenotypes. Next, the study asked if the tissue environment, specifically the amyloid plaques and neurofibrillary tangles characteristic of Alzheimer's disease, has any bearing on microglia's cellular and molecular phenotypes. QUIVER allowed the visualization of five molecular microglia/macrophage phenotypes using digital pathology tools. The recognizable reactive and homeostatic microglia/macrophage phenotypes demonstrated spatial polarization towards and away from amyloid plaques, respectively. Yet, microglia morphology appearance did not always correspond to molecular phenotype. This research not only sheds light on the biology of microglia but also offers QUIVER, a new tool for examining pathological alterations in the brains of the elderly.© 2023. The Author(s).