Protocol for whole-mount immunofluorescence staining of ECM gel-embedded innervated pancreatic organoids.

ECM 凝胶包埋的神经支配的胰腺类器官的整体免疫荧光染色方案。

• 发表时间: 2024 Jun 21
• 来源期刊: STAR Protoc
• DOI: 10.1016/j.xpro.2024.103132
• PMID: 38875112
• 文章类型: Journal Article
• 作者列表: Beşikcioğlu HE,Yurteri Ü,Ye L,Zhang F,Moretti A,Gürcinar IH,Dogruöz A,Karakas D,Friess H,Ceyhan GO,Istvanffy R,Ekin Demir I

关键词: Microscopy; Model Organisms; Organoids

Mesh: Organoids / cytology metabolism; Extracellular Matrix / metabolism chemistry; Pancreas / cytology metabolism; Fluorescent Antibody Technique / methods; Animals; Staining and Labeling / methods; Humans; Gels / chemistry; Mice

Abstract

The mandatory usage of extracellular matrix (ECM) gels in 3D cultures limits antibody penetration and increases background, while the removal of ECM gel causes disruption of morphology and sample loss. These factors pose challenges to effective immune labeling-based staining. Here, we present a protocol for whole-mount immunofluorescence staining of gel-embedded pancreatic organoids. We describe steps for sample fixation, blocking, and antibody incubation. We detail procedures for washing antibodies and mounting.

摘要

在3D培养物中强制使用细胞外基质（ECM）凝胶限制了抗体渗透并增加了背景，而ECM凝胶的去除导致形态破坏和样品损失。这些因素对有效的基于免疫标记的染色提出了挑战。这里,我们提出了一种对凝胶包埋的胰腺类器官进行整体免疫荧光染色的方案。我们描述了样本固定的步骤，阻塞,和抗体孵育。我们详细介绍了清洗抗体和安装的程序。