Immunofluorescence staining is a crucial tool for studying the structure and behavior of intracellular proteins and organelles. During the staining process, the permeabilization treatment is usually required to enhance the penetration of a fluorescent antibody into the cells. However, since most of the membrane imaging dyes as well as the membrane lipids will detach from the cell surface after permeabilization, membrane labeling using these dyes is not compatible with immunofluorescence staining. Herein, by linking cholesterol-polyethylene glycol (PEG-Chol) and fluorescein isothiocyanate (FITC) with the amine-rich glycol chitosan (GC), we prepared a multifunctional polymeric construct, GC-PEG Chol-FITC, and realized permeabilization-tolerant plasma membrane imaging. Owing to the presence of abundant amine groups in the labeling reagent and the membrane proteins/lipids, the addition of paraformaldehyde in the fixation step induces the amine-cross-linking between the labeling reagents and the membrane proteins/lipids, thus preventing the detachment of fluorophores from the cell surface after permeabilization. Besides, the large molecular weight effect of the imaging reagent may also account for its antipermeabilization property. Furthermore, by combining immunofluorescence staining with the plasma membrane labeling by GC-PEG Chol-FITC, we simultaneously imaged the plasma membrane and cytoskeletons, and clearly observed metaphase cells and binucleated cells. The concept of using amine-rich polymeric dyes for plasma membrane imaging will inspire the development of more permeabilization-resistant membrane labeling dyes with better performance, which can realize simultaneous membrane and intracellular protein imaging and facilitate the future studies of membrane–intracellular protein interactions.

中文翻译：

---

富胺乙二醇壳聚糖衍生物的耐透性血浆膜成像试剂

免疫荧光染色是研究细胞内蛋白质和细胞器的结构和行为的重要工具。在染色过程中，通常需要透化处理以增强荧光抗体向细胞的渗透。然而，由于透化后大多数膜成像染料以及膜脂质将从细胞表面脱离，因此使用这些染料进行膜标记与免疫荧光染色不兼容。本文中，通过将胆固醇-聚乙二醇（PEG-Chol）和异硫氰酸荧光素（FITC）与富含胺的乙二醇壳聚糖（GC）连接，我们制备了多功能聚合物构建体GC-PEG Chol-FITC，并实现了耐渗透性的血浆膜成像。由于标记试剂和膜蛋白/脂质中存在丰富的胺基，因此在固定步骤中添加多聚甲醛会引起标记试剂和膜蛋白/脂质之间的胺交联，从而防止了脂质的分离。透化后来自细胞表面的荧光团。此外，显像剂的大分子量效应也可以解释其抗通透性。此外，通过结合免疫荧光染色和GC-PEG Chol-FITC标记的质膜，我们同时对质膜和细胞骨架成像，并清晰观察到中期细胞和双核细胞。