Shihan Mahbubul H, Novo Samuel G, Le Marchand Sylvain J, Wang Yan, Duncan Melinda K
Department of Biological Sciences, University of Delaware, Newark, DE, 19716, USA.
Delaware Biotechnology Institute, Bioimaging Center, University of Delaware, Newark, DE, 19713, USA.
Biochem Biophys Rep. 2021 Feb 1;25:100916. doi: 10.1016/j.bbrep.2021.100916. eCollection 2021 Mar.
Western blotting (WB), enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC) have long been used to assess and quantitate relative protein expression in cultured cells and tissue samples. However, WB and ELISA have limited ability to meaningfully quantitate relative protein levels in tissues with complex cell composition, while tissue dissociation followed by FC is not feasible when tissue is limiting and/or cells difficult to isolate. While protein detection in tissue using immunofluorescent (IF) probes has traditionally been considered a qualitative technique, advances in probe stability and confocal imaging allow IF data to be easily quantitated, although reproducible quantitation of relative protein expression requires careful attention to appropriate controls, experiment design, and data collection. Here we describe the methods used to quantify the data presented in et al. which lays out a workflow where IF data collected on a confocal microscope can be used to quantitate the relative levels of a molecule of interest by measuring mean fluorescent intensity across a region of interest, cell number, and the percentage of cells in a sample "positive" for staining with the fluorescent probe of interest. Overall, this manuscript discusses considerations for collecting quantifiable fluorescent images on a confocal microscope and provides explicit methods for quantitating IF data using FIJI-ImageJ.
蛋白质免疫印迹法（WB）、酶联免疫吸附测定法（ELISA）和流式细胞术（FC）长期以来一直用于评估和定量培养细胞及组织样本中的相对蛋白质表达。然而，WB和ELISA在对细胞组成复杂的组织中的相对蛋白质水平进行有意义的定量方面能力有限，而当组织有限和/或细胞难以分离时，先进行组织解离再进行FC分析是不可行的。虽然传统上使用免疫荧光（IF）探针检测组织中的蛋白质被认为是一种定性技术，但随着探针稳定性和共聚焦成像技术的进步，IF数据能够轻松实现定量分析，不过要对相对蛋白质表达进行可重复的定量分析，需要仔细关注适当的对照、实验设计和数据收集。在此，我们描述了用于量化等人文章中所呈现数据的方法，该文章阐述了一种工作流程，即通过在共聚焦显微镜上收集的IF数据，测量感兴趣区域的平均荧光强度、细胞数量以及样本中被感兴趣的荧光探针“阳性”染色的细胞百分比，从而定量感兴趣分子的相对水平。总体而言，本文讨论了在共聚焦显微镜上收集可量化荧光图像的注意事项，并提供了使用FIJI-ImageJ定量IF数据的具体方法。
