Cryosectioning and Immunohistochemistry Using Frozen Adult Murine Brain Neural Tissue.

Cryopreservation and immunohistochemistry offer a comprehensive, robust, and simple methodology to investigate neural patterning and cellular function. Rapid freezing of the whole brain allows excellent preservation of neural ultrastructure and tissue architecture without destroying sensitive protein epitopes that are often compromised following standard paraffin embedding histological techniques. Here, we present a rapid and simple protocol for employing cryosectioning and subsequent immunohistochemistry in the study of adult murine brain neural tissue.

Microscopic Imaging to Visualize the Distribution of Dietary Nucleic Acids in Food Products of Various Origins.

Dietary nucleic acids (dietNAs) are being increasingly recognized as important food components with nutritional value. However, the precise dietary recommendations for dietNAs are limited, because established methods for determining the quantity and nutritional role of dietNAs are still lacking. One of the tools to narrow this gap could be microscopic imaging, as a convenient approach to visualize the abundance and distribution of dietNAs in food products. With the aid of appropriate bioinformatic elaboration, such images may in future enable the direct semiquantitative estimation of these macromolecules in food products. In the presented study, two methods of preparing microscopic sections and staining them with DNA-specific fluorochromes were used for microscopic imaging of dietNAs in food products of plant and animal origin. Procedures for preparing formalin-fixed paraffin-embedded sections and cryosections were compared in terms of their usefulness for routine food analysis. Both methods turned out equally suitable for visualizing dietNA distribution in animal and plant products. However, the use of cryosections allowed a significantly shorter analysis time and reduced the consumption of organic solvents. Both of these advantages make the cryosection method preferable while establishing a dedicated methodology for routine assessment of dietNAs in the food industry.

Cryosectioning of Hydrogels as a Reliable Approach to Increase Yield and Further Tune Mechanical Properties.

Decellularized extracellular matrix (dECM) hydrogels have emerged as promising materials in tissue engineering. The steps to produce dECM hydrogels containing the bioactive epitopes found in the native matrix are often laborious, including the initial harvesting and decellularization of the animal organ. Furthermore, resulting hydrogels often exhibit weak mechanical properties that require the use of additional crosslinkers such as genipin to truly simulate the mechanical properties of the desired study tissue. In this work, we have developed a protocol to readily obtain tens of thin dECM hydrogel cryosections attached to a glass slide as support, to serve as scaffolds for two-dimensional (2D) or three-dimensional (3D) cell culture. Following extensive atomic force microscopy (AFM)-based mechanical characterization of dECM hydrogels crosslinked with increasing genipin concentrations (5 mM, 10 mM, and 20 mM), we provide detailed protocol recommendations for achieving dECM hydrogels of any biologically relevant stiffness. Given that our protocol requires hydrogel freezing, we also confirm that the approach taken can be further used to increase the mechanical properties of the scaffold in a controlled manner exhibiting twice the stiffness in highly crosslinked arrays. Finally, we explored the effect of ethanol-based short- and long-term sterilization on dECM hydrogels, showing that in some situations it may give rise to significant changes in hydrogel mechanical properties that need to be taken into account in experimental design. The hydrogel cryosections produced were shown to be biocompatible and support cell attachment and spreading for at least 72 h in culture. In brief, our proposed method may provide several advantages for tissue engineering: (1) easy availability and reduction in preparation time, (2) increase in the total hydrogel volume eventually used for experiments being able to obtain 15-22 slides from a 250 µL hydrogel) with a (3) reduction in scaffold variability (only a 17.5 ± 9.5% intraslide variability provided by the method), and (4) compatibility with live-cell imaging techniques or further cell characterization of cells.

Advancements in 3D spheroid imaging: Optimised cryosectioning and immunostaining techniques.

This article presents a modified protocol for embedding and sectioning spheroids and organoids, which are increasingly used in research due to their ability to emulate living tissue. The modifications aim to reduce the distortion and damage of these fragile structures during the embedding and sectioning process. The new method involves using optimized embedding containers, a modified embedding protocol, and optimized temperatures for cryosectioning. A heat-induced antigen retrieval protocol was tested and found to significantly increase immunostaining intensity without compromising spheroid integrity. The combined approach allowed for the creation of thinner cryosections, leading to clearer and more detailed images. The results suggest that the modified protocol could be widely adopted to enhance the imaging of spheroids and organoids.•Paraformaldehyde fixation of spheroids•Antigen retrieval treatment of spheroids•Embedding in freezing medium and cryosectioning.

Genome-Wide RNA Tomography in the Mouse Whole Olfactory Mucosa.

Spatial transcriptomics allows for the genome-wide profiling of topographic gene expression patterns within a tissue of interest. Here, we describe our methodology to generate high-quality RNA-seq libraries from cryosections from fresh frozen mouse whole olfactory mucosae. This methodology can be extended to virtually any vertebrate organ or tissue sample.

Elucidating the spatial distribution of organic contaminants and their biotransformation products in amphipod tissue by MALDI- and DESI-MS-imaging.

The application of mass spectrometry imaging (MSI) is a promising tool to analyze the spatial distribution of organic contaminants in organisms and thereby improve the understanding of toxicokinetic and toxicodynamic processes. MSI is a common method in medical research but has been rarely applied in environmental science. In the present study, the suitability of MSI to assess the spatial distribution of organic contaminants and their biotransformation products (BTPs) in the aquatic invertebrate key species Gammarus pulex was studied. Gammarids were exposed to a mixture of common organic contaminants (carbamazepine, citalopram, cyprodinil, efavirenz, fluopyram and terbutryn). The distribution of the parent compounds and their BTPs in the organisms was analyzed by two MSI methods (MALDI- and DESI-HRMSI) after cryo-sectioning, and by LC-HRMS/MS after dissection into different organ compartments. The spatial distribution of contaminats in gammarid tissue could be successfully analyzed by the different analytical methods. The intestinal system was identified as the main site of biotransformation, possibly due to the presence of biotransforming enzymes. LC-HRMS/MS was more sensitive and provided higher confidence in BTP identification due to chromatographic separation and MS/MS. DESI was found to be the more sensitive MSI method for the analyzed contaminants, whereas additional biomarkers were found using MALDI. The results demonstrate the suitability of MSI for investigations on the spatial distribution of accumulated organic contaminants. However, both MSI methods required high exposure concentrations. Further improvements of ionization methods would be needed to address environmentally relevant concentrations.

High pressure freezing and cryo-sectioning can be used for protein structure determination by electron diffraction.

Electron diffraction of three-dimensional nanometer sized crystals has emerged since 2013 as an efficient technique to solve the structure of both small organic molecules and model proteins. However, the major bottleneck of the technique when applied to protein samples is to produce nano-crystals that do not exceed 200 to 300 nm in at least one dimension and to deposit them on a grid while keeping the minimum amount of solvent around them. Since the presence of amorphous solvent around the crystal, necessary to preserve its integrity, increases the amount of diffuse scattering, thus degrading the signal-to noise ratio of the diffraction signal, other sample preparation strategies have been developed. One of them is the milling of thin crystal lamella using focused ion beam (FIB), which was successfully applied to several protein crystals. Here, we present a new approach that uses cryo-sectioning after high pressure freezing of dextran embedded protein crystals. 150 to 200 nm thick cryo-sections of hen egg white lysozyme tetragonal crystals where used for electron diffraction experiments. Complete diffraction data up to 2.9 Å resolution have been collected and the lysozyme structure has been solved by molecular replacement and refined against these data. Our data demonstrate that cryo-sectioning can preserve protein structure at high resolution and can be used as a new sample preparation technique for 3D electron diffraction experiments of protein crystals. The different orientations found in the crystal chips and their large number resulting from the cryo-sectioning make the latter an attractive approach as it combines advantages from both blotting approaches (number of crystals) and FIB-milling (controlled thickness and absence of solvent around the crystal).

Development of an UHPLC-MS/MS method to determine cutaneous biodistribution of cannabidiol after topical application of cannabidiol gel assisted by iontophoresis.

Cannabidiol has potential for use in skin disease therapy, so it is important to know the cutaneous biodistribution of cannabidiol after topical application of cannabidiol formulations. However, currently existing quantification methods for the investigation of cannabidiol skin distribution are not optimal. This study aimed to establish a method for the determination of cannabidiol in skin samples by UHPLC-MS/MS. A BEH C18 (50.0 × 2.1 mm, 2.5 µm) column was used; the mobile phase consisted of acetonitrile-0.1% formic acid (70:30, v/v), the flow rate was 0.2 µl·min-1 and the column temperature was 30°C. Positive-ion mode with multiple reaction monitoring detection was used to quantify cannabidiol (m/z 315.1 → 193.1) while diphenhydramine (m/z 256.3 → 167.08) served as the internal standard. Good linearity was shown in the range of 1-200 ng·ml-1 for cannabidiol with correlation coefficients of >0.999. The LLOQ was 1 ng·ml-1 . The intra-day and inter-day RSDs of cannabidiol were all <2%. A cryo-sectioning technique combined with the UHPLC-MS/MS method was used to successfully determine cannabidiol levels in a series of very thin skin layers.

Sample Preparation for Metabolite Detection in Mass Spectrometry Imaging.

Metabolites reflect the biological state of cells and tissue, and metabolomics is therefore a field of high interest both to understand normal physiological functions and disease development. When studying heterogeneous tissue samples, mass spectrometry imaging (MSI) is a valuable tool as it conserves the spatial distribution of analytes on tissue sections. A large proportion of metabolites are, however, small and polar, making them vulnerable to delocalizing through diffusion during sample preparation. Here we present a sample preparation method optimized to limit diffusion and delocalization of small polar metabolites in fresh frozen tissue sections. This sample preparation protocol includes cryosectioning, vacuum frozen storage, and matrix application. The methods described were primely developed for matrix-assisted laser desorption/ionization (MALDI) MSI, but the protocol describing cryosectioning and vacuum freezing storage can also be applied before desorption electrospray ionization (DESI) MSI. Our vacuum drying and vacuum packing approach offers a particular advantage to limit delocalization and safe storage.

Measurement of an evaporation coefficient in tissue sections as a correction factor for 10B determination.

Boron neutron capture therapy (BNCT) is a cancer treatment option that combines preferential uptake of a boron compound in tumors and irradiation with thermal neutrons. For treatment planning, the boron concentration in different tissues must be considered. Neutron autoradiography using nuclear track detectors (NTD) can be applied to study both the concentration and microdistribution of boron in tissue samples. Histological sections are obtained from frozen tissue by cryosectioning. When the samples reach room temperature, they undergo an evaporation process, which leads to an increase in the boron concentration. To take this effect into account, certain correction factors (evaporation coefficients, CEv) must be applied. With this aim, a protocol was established to register and analyze mass variation of tissue sections, measured with a semimicro scale. Values of ambient temperature, pressure, and humidity were simultaneously recorded. Reproducible results of evaporation curves and CEv values were obtained for different tissue samples, which allowed the systematization of the procedure. This study could contribute to a more precise determination of boron concentration in tissue samples through the neutron autoradiography technique, which is of great relevance to make dosimetric calculations in BNCT.

A quick and laidback way to detect the internal structure of the Drosophila eye: An alternative to cryosectioning.

Immunofluorescence techniques have been a great tool to chase the structure, localization, and function of many proteins within a cell. Drosophila eye is widely used as a model to answer various questions. However, the complex sample preparation and visualization methods restrict its use only with an expert's hand. Thus, an easy and hassle-free method is in need to broaden the use of this model even with an amateur's hand. The current protocol describes an easy sample preparation method using DMSO to image the adult fly eye. The brief description of sample collection, preparation, dissection, staining, imaging, storage, and handling has been described over here. For readers, the possible problems that might arise during the execution of the experiment have been described with their possible reason and solutions. The overall protocol reduces the use of chemicals and shortens the sample preparation time to only 3 h, which is significantly less in comparison to other protocols.

Laser Microdissection: A High-Precision Approach to Isolate Specific Cell Types from Any Plant Species for Downstream Molecular Analyses.

Plants display a great diversity of particular cell types that obviously perform functions and regulations that are essential for successful growth and development, whether under optimal or adverse conditions. The functions performed by each of these particular cell types must be associated with specific transcriptomic, proteomic, and metabolic profiles that cannot be disentangled by analyzing whole plant organs and tissues. Laser microdissection is a technique for the collection of specific cell types in plant organs and tissues comprising heterogeneous cell populations. It has been successfully used for physiological and molecular studies. Laser microdissection can be applied to any plant species as long as it is possible to reliably identify the cell types of interest. Here, we describe step by step, using citrus as a model plant, a fast, simple, easy to perform, and experimentally validated protocol to collect cells from the abscission zone, a specific tissue that is difficult to access and whose activity is important in the response of plants to adverse environmental conditions.

Atomic Force Microscopy Micro-Indentation Methods for Determining the Elastic Modulus of Murine Articular Cartilage.

The mechanical properties of biological tissues influence their function and can predict degenerative conditions before gross histological or physiological changes are detectable. This is especially true for structural tissues such as articular cartilage, which has a primarily mechanical function that declines after injury and in the early stages of osteoarthritis. While atomic force microscopy (AFM) has been used to test the elastic modulus of articular cartilage before, there is no agreement or consistency in methodologies reported. For murine articular cartilage, methods differ in two major ways: experimental parameter selection and sample preparation. Experimental parameters that affect AFM results include indentation force and cantilever stiffness; these are dependent on the tip, sample, and instrument used. The aim of this project was to optimize these experimental parameters to measure murine articular cartilage elastic modulus by AFM micro-indentation. We first investigated the effects of experimental parameters on a control material, polydimethylsiloxane gel (PDMS), which has an elastic modulus on the same order of magnitude as articular cartilage. Experimental parameters were narrowed on this control material, and then finalized on wildtype C57BL/6J murine articular cartilage samples that were prepared with a novel technique that allows for cryosectioning of epiphyseal segments of articular cartilage and long bones without decalcification. This technique facilitates precise localization of AFM measurements on the murine articular cartilage matrix and eliminates the need to separate cartilage from underlying bone tissues, which can be challenging in murine bones because of their small size. Together, the new sample preparation method and optimized experimental parameters provide a reliable standard operating procedure to measure microscale variations in the elastic modulus of murine articular cartilage.

Ex-vivo Skin Permeability Tests of Nanoparticles for Microscopy Imaging.

Delivery of drugs through the skin is a major challenge in the field of drug delivery systems. Quantification of materials, and specifically nanoparticles, within the skin layers is essential for the development of advanced topical and transdermal delivery systems. We have developed a technique for ex-vivo segmentation and evaluation of human skin samples treated with fluorescent nanoparticles. The method is based on horizontal cryosections of skin samples, followed by confocal microscopy and image analysis. This protocol is relatively simple to perform with basic histological tools, thus it can serve for various dermatology assays.

Spatially Mapping the Baseline and Bisphenol-A Exposed Daphnia magna Lipidome Using Desorption Electrospray Ionization-Mass Spectrometry.

Untargeted lipidomics has previously been applied to the study of daphnids and the discovery of biomarkers that are indicative of toxicity. Typically, liquid chromatography-mass spectrometry is used to measure the changes in lipid abundance in whole-body homogenates of daphnids, each only ca. 3 mm in length which limits any biochemical interpretation of site-specific toxicity. Here, we applied mass spectrometry imaging of Daphnia magna to combine untargeted lipidomics with spatial resolution to map the molecular perturbations to defined anatomical regions. A desorption electrospray ionization-mass spectrometry (DESI-MS) method was optimized and applied to tissue sections of daphnids exposed to bisphenol-A (BPA) compared to unexposed controls, generating an untargeted mass spectrum at each pixel (35 µm2/pixel) within each section. First, unique lipid profiles from distinct tissue types were identified in whole-body daphnids using principal component analysis, specifically distinguishing appendages, eggs, eye, and gut. Second, changes in the lipidome were mapped over four stages of normal egg development and then the effect of BPA exposure on the egg lipidome was characterized. The primary perturbations to the lipidome were annotated as triacylglycerides and phosphatidylcholine, and the distributions of the individual lipid species within these classes were visualized in whole-body D. magna sections as ion images. Using an optimized DESI-MS workflow, the first ion images of D. magna tissue sections were generated, mapping both their baseline and BPA-perturbed lipidomes.

Cryosectioning.

The protocols presented here describe steps for cryosectioning tissue samples to be used in light microscopy methodologies including histochemistry, enzyme immunohistochemistry, and immunofluorescence. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Cryosectioning.

Intercellular nuclear migration in cryofixed tobacco male meiocytes.

In this study, intercellular nuclear migration (INM), also known as cytomixis, was documented in cryofixed plant meiocytes for the first time. Intact tobacco inflorescences and flower buds as well as dissected individual anthers were cryofixed in liquid nitrogen by plunge freezing. Cryosubstituted and cryosectioned male meiocytes were analyzed by light microscopy. For cryosubstitution, the frozen material was kept in acetic alcohol at - 70 °C for 1 week. For cryosectioning, the frozen material was sectioned at - 20 °C, and fixed with precooled acetic alcohol. Fixation of the intact tobacco inflorescences in Carnoy's solution was used as a control. Microscopy revealed good preservation of cell structure in the cryofixed anthers, flower buds, and inflorescences. INM was detectable in all the studied cryofixed and chemically fixed samples. The cytological picture of INM observed in the cryofixed meiocytes did not noticeably differ from the picture obtained with the chemically fixed cells. These results indicate that INM is observable irrespective of whether a physical or chemical fixation method is employed, with minimal damage from handling. Our results contradict the notion that INM is a phenomenon caused by mechanical, osmotic, or chemical artifacts during sample preparation.

Tissue Analysis of Lung-Targeted Delivery of siRNA and Plasmid DNA.

Development of nucleic acid delivery systems requires the use of adequate tissue analysis methods, especially when aiming tissue-targeted drug delivery. In this chapter, a protocol is presented for analyzing a reporter signal from the lung tissue. Because lung is an important target tissue from the clinical point of view, yet represents a challenge from the histological point of view, this protocol can be used in any lung-targeting drug delivery project.

High-throughput imaging of fresh-frozen plant reproductive samples in a variable pressure SEM.

Conventional light and electron microscopy are the most widely used techniques for examining plant reproductive tissues; however, they are time-consuming or expensive. The anther is the male part of the plant reproductive system. Structural changes drive development, and any structural defect may lead to an increase in fertility or cause sterility; thus, quick detection of structural changes is crucial in reproductive biology. We optimized an existing low-temperature SEM alternative to examine the internal structure of hydrated, fresh-frozen anthers. In contrast with the original technique, our method does not require precooling adhesion (ethanol to fix the specimen), and the cryo-sectioning can be conducted at atmospheric pressure. In addition to enabling the differentiation between aerial and liquid-filled intercellular spaces, this method is expected to facilitate the detection of quick (during a day) developmental changes in plant reproductive tissues, which is a current challenge using conventional approaches.•This method allows the high-throughput imaging of fresh-frozen plant reproductive samples collected every 10 min, which is important for developmental studies.•The cryo-images of samples with thickness ranging from 0.2 to 3 mm can be well-preserved at 800X magnification.•This method does not require chemical processing, critical point drying, customized cryo-accessories, controlled temperature cold stages, or metal coating. This simplified method does not require highly skilled personnel, and it is suitable in most microscopy laboratories.

Differential Tropism in Roots and Shoots of Resistant and Susceptible Cassava (Manihot esculenta Crantz) Infected by Cassava Brown Streak Viruses.

Cassava brown streak disease (CBSD) is a destructive disease of cassava in Eastern and Central Africa. Because there was no source of resistance in African varieties to provide complete protection against the viruses causing the disease, we searched in South American germplasm and identified cassava lines that did not become infected with the cassava brown streak viruses. These findings motivated further investigations into the mechanism of virus resistance. We used RNAscope® in situ hybridization to localize cassava brown streak virus in cassava germplasm lines that were highly resistant (DSC 167, immune) or that restricted virus infections to stems and roots only (DSC 260). We show that the resistance in those lines is not a restriction of long-distance movement but due to preventing virus unloading from the phloem into parenchyma cells for replication, thus restricting the virus to the phloem cells only. When DSC 167 and DSC 260 were compared for virus invasion, only a low CBSV signal was found in phloem tissue of DSC 167, indicating that there is no replication in this host, while the presence of intense hybridization signals in the phloem of DSC 260 provided evidence for virus replication in companion cells. In neither of the two lines studied was there evidence of virus replication outside the phloem tissues. Thus, we conclude that in resistant cassava lines, CBSV is confined to the phloem tissues only, in which virus replication can still take place or is arrested.