使用Opal-TSA染料进行信号放大的肺结核病变三维多重荧光成像方案。
Protocol for 3D multiplexed fluorescent imaging of pulmonary TB lesions using Opal-TSA dyes for signal amplification.
作者信息
Lata Suruchi, Yabaji Shivraj M, O'Connell Aoife K, Gertje Hans P, Kirber Michael T, Crossland Nicholas A, Kramnik Igor
机构信息
The National Emerging Infectious Diseases Laboratories (NEIDL), Boston University, Boston, MA, USA.
The National Emerging Infectious Diseases Laboratories (NEIDL), Boston University, Boston, MA, USA; The Department of Pathology and Laboratory Medicine, Boston University Chobanian & Avedisian School of Medicine, Boston, MA, USA.
出版信息
STAR Protoc. 2025 Mar 21;6(1):103640. doi: 10.1016/j.xpro.2025.103640. Epub 2025 Feb 20.
To detect multiple protein markers, here we present a protocol for imaging pulmonary tuberculosis (PTB) lesions from murine lungs using Opal-tyramide signal amplification (TSA) conjugated fluorescent dyes in free-floating formalin-fixed thick lung sections (50-100 μm). We describe steps for preparing tissue sections, permeabilization and antigen retrieval, and multiplexing with antibodies raised in the same species. This protocol has been optimized to preserve tissue integrity and endogenously expressed fluorescent reporter signals and nuclear staining. It enhances the signal-to-background ratio and enables 3D image rendering.
为了检测多种蛋白质标志物，我们在此介绍一种方案，用于在自由漂浮的福尔马林固定厚肺切片（50 - 100μm）中，使用与荧光染料偶联的Opal酪胺信号放大（TSA）对小鼠肺部的肺结核（PTB）病变进行成像。我们描述了制备组织切片、通透化和抗原修复的步骤，以及与同种动物产生的抗体进行多重检测的步骤。该方案经过优化，以保留组织完整性、内源性表达的荧光报告信号和核染色。它提高了信噪比，并能够进行三维图像渲染。
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