A rapid and convenient method for fluorescence analysis of in vitro cultivated metacestode vesicles from Echinococcus multilocularis.

Zhe Cheng,Yanhai Wang,Fan Liu,Huimin Tian,Liang Wang,Shan Zhu,Jinxing Lin

doi:10.1371/journal.pone.0118215

Abstract

We here describe a convenient method for preparation, fixation and fluorescence analysis of in vitro cultivated metacestode vesicles from E. multilocularis. Parasite materials could be prepared in one hour, did not need to be sectioned, and were subsequently utilized for further whole-mount staining assays directly. Using these preparations, in combination with conventional fluorescence staining techniques, we could detect the expression and subcellular localization of a specific protein and identify in situ proliferative or apoptotic cells in the germinal layer of metacestode vesicles. Based on this approach, future molecular and cellular analysis of Echinococcus metacestode vesicles in the in vitro system will be greatly facilitated.

Highlights

The metacestode larval stage of the fox-tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), which is considered to be one of the most lethal helminthic infections in humans [1]
The metacestode vesicles are bounded by a thin cellular layer and filled by hydatid cyst fluid
The in vitro cultivated vesicles were washed in phosphate buffer saline (PBS), manually picked up and mounted onto a poly-L-lysine coated glass slide with pipettes

Summary

Introduction

The metacestode larval stage of the fox-tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), which is considered to be one of the most lethal helminthic infections in humans [1]. A Method for Fluorescence Analysis of E. multilocularis Vesicles Klaus Brehm’s research group has successfully developed an effective method for whole-mount immunofluorescence analysis of in vitro cultivated E. multilocularis vesicles [5, 6]. Simple and rapid sample preparation methods that facilitate the staining of protein / nucleic acid in the GL of vesicles are still needed.

Results

Conclusion