Development of a Plastic Embedding Method for Large-Volume and Fluorescent-Protein-Expressing Tissues

Abstract

Fluorescent proteins serve as important biomarkers for visualizing both subcellular organelles in living cells and structural and functional details in large-volume tissues or organs. However, current techniques for plastic embedding are limited in their ability to preserve fluorescence while remaining suitable for micro-optical sectioning tomography of large-volume samples. In this study, we quantitatively evaluated the fluorescence preservation and penetration time of several commonly used resins in a Thy1-eYFP-H transgenic whole mouse brain, including glycol methacrylate (GMA), LR White, hydroxypropyl methacrylate (HPMA) and Unicryl. We found that HMPA embedding doubled the eYFP fluorescence intensity but required long durations of incubation for whole brain penetration. GMA, Unicryl and LR White each penetrated the brain rapidly but also led to variable quenching of eYFP fluorescence. Among the fast-penetrating resins, GMA preserved fluorescence better than LR White and Unicryl. We found that we could optimize the GMA formulation by reducing the polymerization temperature, removing 4-methoxyphenol and adjusting the pH of the resin solution to be alkaline. By optimizing the GMA formulation, we increased percentage of eYFP fluorescence preservation in GMA-embedded brains nearly two-fold. These results suggest that modified GMA is suitable for embedding large-volume tissues such as whole mouse brain and provide a novel approach for visualizing brain-wide networks.

Plastic embedding immunolabeled large-volume samples for three-dimensional high-resolution imaging

“…Our results showed that the fluorophores could be preserved better in Lowicryl HM20 and GMA, whereas CF568 and CY5 fluorophores degraded more severely in LR White. This decrease might be related to the relatively acid environment in LR White [35]. Considering the cutting performance [39], we chose Lowicryl HM20 resin to embed the IF stained sample.…”

“…The acrylic resin is optimal for fluorescent imaging considering the viscosity and autofluorescence [33,34]. So, we chose three types of acrylic resins, including hydrophilic and non-cross-linking resin (GMA), hydrophilic and cross-linking resin (LR-White), and hydrophobic and cross-linking resin (Lowicryl HM20) as the embedding medium, and used them to embed IF-stained samples with the optimized embedding procedure [24,35,36]. We labeled 100-μm-thick mouse brain tissue with a primary antibody anti-tyrosine hydroxylase, while secondary antibodies were conjugated with four different fluorescent probes: CF488, CF568, CY3, and CY5 dyes (Tables 1 and 2); and the brain slice images are shown in Fig.…”

High axial resolution imaging system for large volume tissues using combination of inclined selective plane illumination and mechanical sectioning

“…The sample is embedded in resin for mechanical sectioning to reach an extended range in axial scale. With prior researches about plastic embedding techniques, fluorescence in tissues could be well maintained for high resolution imaging [23][24][25]. To restrain aberrations increased by plastic embedding, a thin layer of the sample surface will be imaged during one imaging process.…”

High-refractive index of acrylate embedding resin clarifies mouse brain tissue

“…This method can clear large samples directly without requiring special pretreatment, and can maintain the endogenous fluorescent signal well. In this study, we investigated the effect of differences in the pH value of monomers on green fluorescent protein (GFP) fluorescence, 23 and a transparent GFP mouse brain block imaged via light-sheet fluorescence microscopy.…”

“…23 Thus, the pH value of the resin monomer can affect endogenous GFP signal intensity markedly. In our study, solution c was the best for maintaining the GFP signal.…”