Current methods for in vivo patch-clamp electrophysiology and optical imaging of calcium transients in C. elegans neurons rely on veterinary-grade cyanoacrylate glue for immobilizing animals (Goodman et al., 1998; Lockery and Goodman, 1998; Kerr, et al., 2000). Just about ten years have passed since the first studies measuring cellular responses in individual neurons were published, all of them using Nexaband S/C (WPI, Sarasota, FL) to immobilize worms on thin, 2% agarose pads. Recently and tragically, Abbott discontinued the manufacture of Nexaband S/C. HistoacrylBlue has been used to immobilize animals during recordings from the C. elegans neuromuscular junction (Richmond and Jorgensen, 1999). Unfortunately, the dye added to this adhesive is fluorescent when viewed under a GFP filter set, obscuring GFP-tagged neurons.
Alternative methods for immobilization have emerged from the recent explosion in the design and fabrication of microfluidics devices for worms (Ben-Yakar et al., 2009). These devices have been used for long- and short-term imaging work and appear to be significantly less toxic than even the best cyanoacrylate adhesives. However, the need for physical access to the animals during an electrophysiology experiment precludes the use of existing traps for in vivo recording. Thus, despite the great advances provided by microfluidics devices, there is a continuing need for a minimally toxic, cyanoacrylate adhesive.
We are pleased to report a suitable alternative, custom-formulation from GluStitch Inc. (http://www.glustitch.com, contact: Steven Blacklock, email@example.com): 80% octyl-20% butyl cyanoacrylate (r = 5.75 centiPoise). The viscosity and polymerization rate of this glue is such that it can be applied in a targeted way to one side of a worm without coating the entire animal. With this formulation, it is possible to see the GFP label and also to expose GFP-tagged neurons of interest, the first step needed for electrophysiological recordings. Additionally, worms appear healthy hours after being glued. Other glues and adhesion methods tested were deemed unsuitable. These include: Nexcare (3M), other formulations from GluStitch, Inc. (high viscosity (75 cP) 100% butyl cyanoacrylate, 50/50 octyl/butyl cyanoacrylate, 100% butyl cyanoacrylate (3.7 cP)) and Gluture (Abbott Laboratories). Good luck and good patching.
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