Development of a plastic embedding method for large-volume and fluorescent-protein-expressing tissues

Area of Science:

• Neuroscience
• Biotechnology
• Materials Science

Background:

• Fluorescent proteins are crucial biomarkers for cellular and tissue imaging.
• Existing plastic embedding methods struggle to preserve fluorescence in large samples for micro-optical sectioning tomography.

Purpose of the Study:

• To evaluate and optimize resin embedding techniques for preserving fluorescence in whole mouse brains.
• To identify suitable resins for micro-optical sectioning tomography of large-volume neural tissues.

Main Methods:

• Quantitative evaluation of fluorescence preservation and resin penetration in Thy1-eYFP-H transgenic whole mouse brains.
• Comparison of glycol methacrylate (GMA), LR White, hydroxypropyl methacrylate (HPMA), and Unicryl resins.
• Optimization of GMA formulation by adjusting polymerization temperature, removing 4-methoxyphenol, and altering pH.

Main Results:

• HPMA doubled eYFP fluorescence but had slow penetration.
• GMA, Unicryl, and LR White showed rapid penetration but variable fluorescence quenching.
• Optimized GMA formulation nearly doubled eYFP fluorescence preservation compared to standard GMA.

Conclusions:

• Modified GMA resin offers a viable solution for embedding large-volume tissues like whole mouse brains while preserving fluorescence.
• This optimized embedding technique provides a novel approach for visualizing extensive brain networks.