Our goal was to find an optimal tissue clearing protocol for whole-mount imaging of embryonic and adult hearts and whole embryos of transgenic mice that would preserve green fluorescent protein GFP fluorescence and permit comparison of different currently available 3D imaging modalities. We tested various published organic solvent- or water-based clearing protocols intended to preserve GFP fluorescence in central nervous system: tetrahydrofuran dehydration and dibenzylether protocol (DBE), SCALE, CLARITY, and CUBIC and evaluated their ability to render hearts and whole embryos transparent. DBE clearing protocol did not preserve GFP fluorescence; in addition, DBE caused considerable tissue-shrinking artifacts compared to the gold standard BABB protocol. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The SCALE clearing resulted in sufficient tissue transparency up to ED12.5; at later stages the useful depth of imaging was limited by tissue light scattering. The best method for the cardiac specimens proved to be the CUBIC protocol, which preserved GFP fluorescence well, and cleared the specimens sufficiently even at the adult stages. In addition, CUBIC decolorized the blood and myocardium by removing tissue iron. Good 3D renderings of whole fetal hearts and embryos were obtained with optical projection tomography and selective plane illumination microscopy, although at resolutions lower than with a confocal microscope. Comparison of five tissue clearing protocols and three imaging methods for study of GFP mouse embryos and hearts shows that the optimal method depends on stage and level of detail required.

• Keywords
• Confocal microscopy, Embryo, Green fluorescent protein (GFP), Heart, Optical projection tomography, Tissue transparency,
• MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Heart embryology   MeSH
• Green Fluorescent Proteins analysis   biosynthesis   genetics   MeSH
• Imaging, Three-Dimensional methods   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Green Fluorescent Proteins MeSH

Fibre type determination requires a large series of differently stained muscle sections. The manual identification of individual fibres through the series is tedious and time consuming. This paper presents a software that enables (i) adjusting the position of individual fibres through a series of differently stained sections (image registration) and identification of individual fibres through the series as well as (ii) muscle fibre classification and (iii) quantitative analysis. The data output of the system is the following: numerical and areal proportions of fibre types, fibre type size and optical density (grey level) of the final reaction product in every fibre. The muscle fibre type can be determined stepwise, based on one set of stained sections while further, newly stained sections can be added to the already defined muscle fibre profile. Several advantages of the presented software application in skeletal muscle research are presented. The system is semiquantitative, flexible, and user friendly.

• MeSH
• In Situ Hybridization MeSH
• Immunohistochemistry MeSH
• Muscle Fibers, Skeletal classification   cytology   MeSH
• Rats MeSH
• Humans MeSH
• Masseter Muscle cytology   MeSH
• Myosins genetics   metabolism   MeSH
• Image Processing, Computer-Assisted methods   MeSH
• Protein Isoforms genetics   metabolism   MeSH
• Reproducibility of Results MeSH
• Software * MeSH
• Myosin Heavy Chains genetics   metabolism   MeSH
• User-Computer Interface MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Myosins MeSH
• Protein Isoforms MeSH
• Myosin Heavy Chains MeSH

The aim of this study was to determine whether capillarity in the denervated and reinnervated rat extensor digitorum longus muscle (EDL) is scaled by muscle fiber oxidative potential. We visualized capillaries adjacent to a metabolically defined fiber type and estimated capillarity of fibers with very high oxidative potential (O) vs fibers with very low oxidative potential (G). Capillaries and muscle fiber types were shown by a combined triple immunofluorescent technique and the histochemical method for NADH-tetrazolium reductase. Stacks of images were captured by a confocal microscope. Applying the Ellipse program, fibers were outlined, and the diameter, perimeter, cross-sectional area, length, surface area, and volume within the stack were calculated for both fiber types. Using the Tracer plug-in module, capillaries were traced within the three-dimensional (3D) volume, the length of capillaries adjacent to individual muscle fibers was measured, and the capillary length per fiber length (Lcap/Lfib), surface area (Lcap/Sfib), and volume (Lcap/Vfib) were calculated. Furthermore, capillaries and fibers of both types were visualized in 3D. In all experimental groups, O and G fibers significantly differed in girth, Lcap/Sfib, and Lcap/Vfib, but not in Lcap/Lfib. We conclude that capillarity in the EDL is scaled by muscle fiber size and not by muscle fiber oxidative potential.

• MeSH
• Muscle Denervation MeSH
• Histocytochemistry MeSH
• Capillaries anatomy & histology   MeSH
• Microscopy, Confocal MeSH
• Muscle Fibers, Skeletal metabolism   MeSH
• Muscle, Skeletal blood supply   innervation   metabolism   MeSH
• Rats MeSH
• Oxidation-Reduction MeSH
• Rats, Wistar MeSH
• Imaging, Three-Dimensional MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH