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

Abstract

High-resolution three-dimensional biomolecule distribution information of large samples is essential to understanding their biological structure and function. Here, we proposed a method combining large sample resin embedding with iDISCO immunofluorescence staining to acquire the profile of biomolecules with high spatial resolution. We evaluated the compatibility of plastic embedding with an iDISCO staining technique and found that the fluorophores and the neuronal fine structures could be well preserved in the Lowicryl HM20 resin, and that numerous antibodies and fluorescent tracers worked well upon Lowicryl HM20 resin embedding. Further, using fluorescence MicroOptical sectioning tomography (fMOST) technology combined with ultra-thin slicing and imaging, we were able to image the immunolabeled large-volume tissues with high resolution. Miyawaki, "Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain," Nat. Neurosci. 14(11), 1481-1488 (2011). 11. J. Sharpe, U. Ahlgren, P. Perry, B. Hill, A. Ross, J. Hecksher-Sørensen, R. Baldock, and D. Davidson, "Optical projection tomography as a tool for 3D microscopy and gene expression studies," Science 296 (

Chemical reactivation of fluorescein isothiocyanate immunofluorescence-labeled resin-embedded samples

“…10 Recently, iDISCO immunostaining technique has brought revolutionary breakthrough in en bloc immunofluorescence labeling, 11 and based on it, researchers combined immunofluorescence labeling with resin embedding to visualize three-dimensional (3-D) biomolecule distribution information of immunolabeled large tissues with high resolution. 12 To succeed, they have evaluated the fluorescence behavior of commonly used fluorescent probes in commonly used resins and then screened out the suitable combinations for fluorescence imaging.…”

Multi-Fluorescence High-Resolution Episcopic Microscopy (MF-HREM) for Three-Dimensional Imaging of Adult Murine Organs

“…Various commercially-available resins are used in histology, however as these resins are designed to be manually cut and subsequently stained, they are not optimised for automated, thin sectioning, fluorescence preservation or, in many cases, large samples (28,29). We selected five commercial resins based on the literature which covered the three broad chemical categories for hard resins: methacrylate resins - Technovit 7100, Technovit 8100 and Lowicryl HM20; epoxy resin - Spurr; and arcylic resins - LR White (28,30–32).…”

“…[39] We investigated the compatibility of five commercial resins, which covered the three broad chemical categories for hard resins: methacrylate resins (Technovit 7100, Technovit 8100 and Lowicryl HM20), epoxy resin (Spurr) and acrylic resins (LR White). [39–41] Resins were assessed for the time taken to set, their compatibility with an opacifying agent OB (see section 2.2.1 for further details), for their cut quality and for hardness.…”

Dl-3-N-Butylphthalide Promotes Angiogenesis in an Optimized Model of Transient Ischemic Attack in C57BL/6 Mice

“…Mice were anesthetized and then transcardially perfused with normal saline and 4% PFA in turn. The brains were removed and placed in 4% PFA for 24 h. After embedding in resin ( Gong et al, 2016 ; Gang et al, 2017 ), the whole brain specimens were imaged continuously using the fMOST system (BioMapping 5000; OEBio Inc., Wuhan, China). All the image tiles were obtained with a time-delay integration line scan charge-coupled device ( Zhou YF.…”