Abstract
Visualization of the interaction between parasitic nematodes and their host enables a better understanding of the development of the nematode during the infectious stages of its life cycle and of the effects of host response on nematode integrity in tissues. Appropriate live imaging of these nematode/host interactions, to date has been hindered by the lack of appropriate molecular tools or efficient labeling agents. Here, we present techniques for the live labeling of the nematode parasite Nippostrongylus brasiliensis (N. brasiliensis) that allows visualization of the parasite in the mouse host for up to 24 h. The external sheath can be labeled with CFSE allowing infective larvae to be identified and followed until the stage of exsheathment. The internal labeling of infective parasites can be performed by ingestion of NY microspheres. The worms can continue to be identified for up to 24 h following exsheathment. This should be applicable to other parasitic nematodes.
Keywords: Nippostrongylus brasiliensis, Nematode, Hookworm, Intravital imaging, Microscopy
Materials and Reagents
Equipment
Procedure
If only external labeling of the larvae is required, follow step A1 from the Internal labeling protocol and then proceed with the external labeling protocol.
Representative data
Figure 2. Representative Fluorescent microscopy image showing: Top left, Nippostrongylus larvae stained internally with YO microspheres and stained externally with CFSE, Top right, Nippostrongylus larvae stained internally with YO micro beads and Bottom right, CFSE labeled empty larval sheath
Notes
Two thorough reviews of the basic techniques involved in the preparation, handling and use of N. brasiliensis are Kassai (1982) and Camberis et al. (2003). The protocol outlined above enables reproducible staining of nematodes and their visualization in vivo in tissues for up to 24 h.
Recipes
Acknowledgments
This protocol was adapted and modified from Hawdon and Schad (1990) and from previous work carried out by Professor Weninger’s Immune Imaging Team at Centenary Institute, Australia. We thank the Hugh Green Cytometry Core for confocal microscopy assistance. This work was supported by The Health Research Council of New Zealand and the Marjorie Barclay Trust.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.