神经科学

Recent advancements in tissue-clearing techniques and volumetric imaging have greatly facilitated visualization and quantification of biomolecules, organelles, and cells in intact organs or even entire organisms. Generally, there are two types of clearing methods: hydrophobic and hydrophilic (i.e., clearing with organic or aqueous solvents, respectively). The popular iDISCO approach and its modifications are hydrophobic methods that involve dehydration, delipidation, decolorization (optional), decalcification (optional), and refractive-index (RI) matching steps. Cleared samples are often stored for a relatively long period of time and imaged repeatedly. However, cleared tissues can become opaque over time, which prevents accurate reimaging. We reasoned that the resurgent haziness is likely due to rehydration, residual lipids, and uneven RI deep inside those tissue samples. For rescue, we have developed a simple procedure based on iDISCO. Beginning with a methanol dehydration, samples are delipidated using dichloromethane, followed by RI matching with dibenzyl ether (DBE). This simple method effectively re-clears mouse brains that have turned opaque during months of storage, allowing the user to effectively image immunolabeled samples over longer periods of time.

Key features

• This simple protocol rescues previously cleared tissue that has turned opaque.

• The method does not cause detectable loss of immunofluorescence from previously stained samples.

Graphical overview

Structural and functional changes in vascular networks play a vital role during development, causing or contributing to the pathophysiology of injury and disease. Current methods to trace and image the vasculature in laboratory settings have proven inconsistent, inaccurate, and labor intensive, lacking the inherent three-dimensional structure of vasculature. Here, we provide a robust and highly reproducible method to image and quantify changes in vascular networks down to the capillary level. The method combines vasculature tracing, tissue clearing, and three-dimensional imaging techniques with vessel segmentation using AI-based convolutional reconstruction to rapidly process large, unsectioned tissue specimens throughout the body with high fidelity. The practicality and scalability of our protocol offer application across various fields of biomedical sciences. Obviating the need for sectioning of samples, this method will expedite qualitative and quantitative analyses of vascular networks. Preparation of the fluorescent gel perfusate takes < 30 min per study. Transcardiac perfusion and vasculature tracing takes approximately 20 min, while dissection of tissue samples ranges from 5 to 15 min depending on the tissue of interest. The tissue clearing protocol takes approximately 24–48 h per whole-tissue sample. Lastly, three-dimensional imaging and analysis can be completed in one day. The entire procedure can be carried out by a competent graduate student or experienced technician.

Key features

• This robust and highly reproducible method allows users to image and quantify changes in vascular networks down to the capillary level.

• Three-dimensional imaging techniques with vessel segmentation enable rapid processing of large, unsectioned tissue specimens throughout the body.

• It takes approximately 2–3 days for sample preparation, three-dimensional imaging, and analysis.

• The user-friendly pipeline can be completed by experienced and non-experienced users.

Graphical overview

Molecular characterization of different cell types in rodent brains is a widely used and important approach in neuroscience. Fluorescent detection of transcripts using RNAscope (ACDBio) has quickly became a standard in situ hybridization (ISH) approach. Its sensitivity and specificity allow for the simultaneous detection of between three and forty-eight low abundance mRNAs in single cells (i.e., multiplexing or hiplexing), and, in contrast to other ISH techniques, it is performed in a shorter amount of time. Manual quantification of transcripts is a laborious and time-consuming task even for small portions of a larger tissue section. Herein, we present a protocol for creating high-quality images for quantification of RNAscope-labeled neurons in the rat brain. This protocol uses custom-made scripts within the open-source software QuPath to create an automated workflow for the careful optimization and validation of cell detection parameters. Moreover, we describe a method to derive mRNA signal thresholds using negative controls. This protocol and automated workflow may help scientists to reliably and reproducibly prepare and analyze rodent brain tissue for cell type characterization using RNAscope.

Graphical abstract

The pancreas is a heavily innervated organ, but pancreatic innervation can be challenging to comprehensively assess using conventional histological methods. However, recent advances in whole-mount tissue clearing and 3D rendering techniques have allowed detailed reconstructions of pancreatic innervation. Optical clearing is used to enhance tissue transparency and reduce light scattering, thus eliminating the need to section the tissue. Here, we describe a modified version of the optical tissue clearing protocol iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) optimized for pancreatic innervation and endocrine markers. The protocol takes 13-19 days, depending on tissue size. In addition, we include protocols for imaging using light sheet and confocal microscopes and for 3D segmentation of pancreatic innervation and endocrine cells using Imaris.

Recently developed CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ-hybridization-compatible Tis-sue-hYdrogel) technique renders the tissue transparent by removing lipids in the tissue, while preserving and stabilizing the cellular and subcellular structures. This provides effective penetration of diverse labeling probes, from primary and secondary antibodies to complementary DNA and RNA strands. Followed by high-resolution 3D imaging of neuronal cells and their projections in thick sections, tissue blocks, whole brains, or whole animals, CLARITY allows for superior quantitative analysis of neuronal tissue. Here, we provide our detailed protocol for PACT (Passive Clarity Technique) in brain tissue of diverse species, including human, non-human primate, rodents, and zebrafish. We describe the six principal steps: (1) Tissue fixation and preparation, (2) Passive lipid removal, (3) Immuno-labeling, (4) Optical clearing, (5) Imaging, (6) 3D visualization and quantification.

Tissue clearing techniques are useful for large-scale three-dimensional fluorescence imaging of thick tissues. However, high-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we present a water-based optical clearing and mounting media, SeeDB2, which is designed for high numerical aperture (NA) objective lenses with oil or glycerol immersion. Using quick and simple soaking procedures, the refractive indices of samples can be matched either to that of immersion oil (1.52) or glycerol (1.46), thus minimizing light scattering and spherical aberrations. Fine morphology and various fluorescent proteins are highly preserved during the clearing and imaging process. Our method is useful for the three-dimensional fluorescence imaging of neuronal circuitry at synaptic resolution using confocal and super-resolution microscopy. SeeDB2 is also useful as a mounting media for the super-resolution imaging of fluorescent proteins.