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Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, the European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field. In the present study, we tested an innovative fixative, glyoxal acid-free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open-label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard phosphate-buffered formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local center reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ± 0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ± 0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organs tested. The present clinical performance study, performed following the international standard for performance evaluation of in vitro diagnostic medical devices, highlights the capability of GAF to ensure both structural preservation and diagnostic value of the preparations.

Changes in the femoral and tibial bone tunnel were studied prospectively after arthroscopic ACL reconstruction with quadruple hamstring autograft. To determine whether tunnel enlargement can be decreased by fixing the graft close to the joint line having a stiffer fixation construct we compared “anatomical” (one absorbable interference screw femorally, and bicortical fixation with two absorbable interference screws tibially) and extracortical fixation techniques (Endobutton femorally, and two no. 6 Ethibond sutures over a suture washer tibially). Over a 2‐year period we evaluated 60 patients clinically (IKDC scale, Cincinnati Knee Score, KT‐1000) and radiographically (confirmed by MRI). The operated knee was radiographed immediately postoperatively and 6 and 24 months postoperatively. The femoral and tibial bone tunnel diameter was measured on anteroposterior and lateral images, and the tunnel area was calculated and compared to the initial area calculated from the perioperative drill size. In the “anatomical” group the immediately postoperative bone tunnel area was 75% larger than the initial tunnel area, after 6 months it was increased another 31%, and between 6 and 24 months it remained basically unchanged. In the “extracortical” group there was no significant enlargement immediately postoperatively, but after 6 months it was 65% larger than the initial area of drill and graft size, and between 6 and 24 months it decreased to 47%. There was no correlation between the amount of tunnel enlargement and clinical scores or KT‐1000 measurement. Arthroscopic ACL reconstruction with quadruple hamstring autograft is associated with bone tunnel enlargement. Using a purely extracortical fixation technique thus significantly increased the tibial and femoral tunnel area during the first 6 postoperative months, while it decreased slightly thereafter. The insertion of large interference screws apparently not only compresses the graft in the bone tunnel but also significantly enlarges the bone tunnel itself. The immediate enlargement at the time of the operation is followed by a reduced further enlargement at 6 months and then stabilization. Tunnel widening did not influence clinical outcome over a 2‐year period.

Advances in multiplexed imaging technologies have drastically improved our ability to characterize healthy and diseased tissues at the single-cell level. Co-detection by indexing (CODEX) relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes and has been used so far to simultaneously visualize up to 60 markers in situ. CODEX enables a deep view into the single-cell spatial relationships in tissues and is intended to spur discovery in developmental biology, disease and therapeutic design. Herein, we provide optimized protocols for conjugating purified antibodies to DNA oligonucleotides, validating the conjugation by CODEX staining and executing the CODEX multicycle imaging procedure for both formalin-fixed, paraffin-embedded (FFPE) and fresh-frozen tissues. In addition, we describe basic image processing and data analysis procedures. We apply this approach to an FFPE human tonsil multicycle experiment. The hands-on experimental time for antibody conjugation is ~4.5 h, validation of DNA-conjugated antibodies with CODEX staining takes ~6.5 h and preparation for a CODEX multicycle experiment takes ~8 h. The multicycle imaging and data analysis time depends on the tissue size, number of markers in the panel and computational complexity. This protocol describes co-detection by indexing, a highly multiplexed imaging technology that uses DNA-conjugated antibodies to image up to 60 markers in formalin-fixed, paraffin-embedded and fresh-frozen tissues.

Nuclear processes in real tissues often are significantly different from those in cultured cells. However, immunostaining on tissue sections needs long fixation which masks antigens and, respectively, antigen retrieval which restores antigen accessibility. These treatments affect the immunostaining results and complicate their interpretation. The problem is especially significant for nuclear antigens which often are very sensitive to both fixation and antigen retrieval. We targeted this problem by a study of several histone modifications and nuclear proteins in tissue sections of mouse retina which contains cells with both conventional and unique inverted nuclei. In the latter, the main chromatin classes form separate concentric shells which simplifies evaluation of the signal distribution. We show that as a rule, longer fixation demands longer antigen retrieval time. Nevertheless, antigens are remarkably diverse in this respect and need individual adjustment. We suggest a robust procedure for immunostaining on sections, that is, a method that allows controlling the differences in immunostaining caused by differences in fixation time and antigen retrieval duration, so that immunostaining protocol can be quickly optimized.

Sca l eS is a tissue clearing method for light and electron microscopy featuring stable tissue preservation for immunochemical and genetic labeling of tissue for 3D signal rendering. The technique enables quantitative and reproducible reconstructions of aged and diseased tissue in animal models and patients for high resolution optical pathology. Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ . Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed Sca l eS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. Sca l eS permitted optical reconstructions of aged and diseased brain in Alzheimer's disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer's disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by Sca l eS. These findings suggest that Sca l eS is a simple and reproducible method for accurate visualization of biological tissue.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids’ phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose–glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.This protocol for clearing and high-resolution 3D imaging of entire organoids expressing fluorescence reporters or following immunolabeling enables confocal, super-resolution confocal, multiphoton and light-sheet microscopy to be performed.

The authors describe a chemical approach for imaging deep into fixed brain tissue using Sca l e, a solution that renders biological samples transparent, but preserves fluorescent signals. This technique allows for imaging at unprecedented depth and at subcellular resolution, and makes three-dimensional reconstruction of neural networks possible without serial sectioning. Optical methods for viewing neuronal populations and projections in the intact mammalian brain are needed, but light scattering prevents imaging deep into brain structures. We imaged fixed brain tissue using Sca l e, an aqueous reagent that renders biological samples optically transparent but completely preserves fluorescent signals in the clarified structures. In Sca l e-treated mouse brain, neurons labeled with genetically encoded fluorescent proteins were visualized at an unprecedented depth in millimeter-scale networks and at subcellular resolution. The improved depth and scale of imaging permitted comprehensive three-dimensional reconstructions of cortical, callosal and hippocampal projections whose extent was limited only by the working distance of the objective lenses. In the intact neurogenic niche of the dentate gyrus, Sca l e allowed the quantitation of distances of neural stem cells to blood vessels. Our findings suggest that the Sca l e method will be useful for light microscopy–based connectomics of cellular networks in brain and other tissues.

Formalin-fixed paraffin-embedded (FFPE) tissues constitute a vast and valuable patient material bank for clinical history and follow-up data. It is still challenging to achieve single cell/nucleus RNA (sc/snRNA) profile in FFPE tissues. Here, we develop a droplet-based snRNA sequencing technology (snRandom-seq) for FFPE tissues by capturing full-length total RNAs with random primers. snRandom-seq shows a minor doublet rate (0.3%), a much higher RNA coverage, and detects more non-coding RNAs and nascent RNAs, compared with state-of-art high-throughput scRNA-seq technologies. snRandom-seq detects a median of >3000 genes per nucleus and identifies 25 typical cell types. Moreover, we apply snRandom-seq on a clinical FFPE human liver cancer specimen and reveal an interesting subpopulation of nuclei with high proliferative activity. Our method provides a powerful snRNA-seq platform for clinical FFPE specimens and promises enormous applications in biomedical research. Formalin-fixed paraffin-embedded (FFPE) tissues constitute a vast and valuable patient material bank, but single nucleus RNAseq using such tissues is challenging. Here the authors develop a droplet-based method called snRandom-seq for high-throughput and sensitive single nucleus RNA-seq of FFPE samples.

This protocol describes how to fix, embed, clear and stain excised organs or whole organisms to create optically transparent samples. This versatile protocol is able to process a wide range of sample types for high-resolution imaging. To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.

Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions. For many years, scientists have used chemicals to preserve brain tissue to observe its fine structure using high power microscopes. Korogod et al. now show that these chemicals, or fixatives, cause the tissue to shrink, giving the false impression that the cells are tightly packed together. This has led to misinterpretations of how the brain is structured. For example, components such as the synapse, used by neurons to communicate with each other, are bathed in a watery environment, rather than being tightly enclosed by neighbouring cells as previously thought. Electron microscopy is the only imaging method that is able to see the detailed structure of the nervous system, including synaptic connections. The technique fires a beam of electrons through a sample held in a vacuum and creates images at a higher magnification than light microscopes. However, the electron beam and the vacuum damages live cells and tissues. Therefore, samples must be ‘fixed’ to preserve them before they are imaged with these methods. However, the standard method for fixing brain tissue uses chemical ‘fixatives’, even though these cause shrinkage, and distort the cells. Korogod et al. used an alternative method of fixation—freezing—to better preserve tiny pieces of mouse brain in their natural state. This was achieved with a technique called ‘high pressure freezing’ that combines jets of liquid nitrogen with very high pressures to instantaneously preserve small samples without causing damage through the formation of ice crystals, or any shrinkage and distortion. Once frozen, the samples of mouse brain are encased in resin, and then imaged with the electron microscope. A comparison between the two preservation techniques showed that chemical fixatives remove the watery environment, or extracellular fluid, that surrounds the cells in the brain, squashing them together. The synapses were surrounded by large amounts of extracellular fluid, but cryo fixation also revealed that these sites of communication between neurons also contained many more vesicles—the packets containing the chemicals that pass signals across the synapse. Another type of cell, the glial cell, that supports and helps to maintain neurons, was also strongly distorted by the chemical fixation. These were understood to tightly wrap around synapses, as well as blood vessels, but cryo fixation showed this to be less prominent. This study illustrates that our understanding of how brain's cells are arranged has ignored the effects of the chemicals used to preserve them. Although cryo fixation is only able to preserve tiny samples, it reveals a truer picture of their natural structure, giving scientists a better understanding of how the brain works.