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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.

This technical report describes a method to clear fixed brain tissues while allowing for fluorescent dye tracing and retaining cellular morphology. The authors demonstrate the utility of the technique by obtaining a wiring diagram for sister mitral cells. We report a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. Our method maintained a constant sample volume during the clearing procedure, an important factor for keeping cellular morphology intact, and facilitated the quantitative reconstruction of neuronal circuits. Combined with two-photon microscopy and an optimized objective lens, we were able to image the mouse brain from the dorsal to the ventral side. We used SeeDB to describe the near-complete wiring diagram of sister mitral cells associated with a common glomerulus in the mouse olfactory bulb. We found the diversity of dendrite wiring patterns among sister mitral cells, and our results provide an anatomical basis for non-redundant odor coding by these neurons. Our simple and efficient method is useful for imaging intact morphological architecture at large scales in both the adult and developing brains.

RNA and DNA analyses from paraffin-embedded tissues (PET) are an important diagnostic tool for characterization of a disease, exploring biomarkers and treatment options. Since nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissue are of limited use for molecular analyses due to chemical modifications of biomolecules alternate, formalin-free fixation reagents such as the PAXgene Tissue system are of evolving interest. Furthermore, biomedical research and biomarker development critically relies on using long-term stored PET from medical archives or biobanks to correlate molecular features with long-term disease outcomes. We therefore performed a comparative study to evaluate the effect of long term storage of FFPE and PAXgene Tissue-fixed and paraffin-embedded (PFPE) tissue at different temperatures on nucleic acid stability and usability in PCR. Matched FFPE and PFPE human tissues from routine clinical setting or rat tissues from a highly controlled animal model were stored at room temperature and 4°C, as well as in case of animal tissues frozen at -20°C and -80°C. RNA and DNA were extracted in intervals for up to nine years, and examined for integrity, and usability in quantitative RT-PCR (RT-qPCR) or PCR (qPCR) assays. PET storage at room temperature led to a degradation of nucleic acids which was slowed down by storage at 4°C and prevented by storage at -20°C or -80°C. Degradation was associated with an amplicon length depending decrease of RT-qPCR and qPCR efficiency. Storage at 4°C improved amplifiability in RT-qPCR and qPCR profoundly. Chemically unmodified nucleic acids from PFPE tissue performed superior compared to FFPE tissue, regardless of storage time and temperature in both human and rat tissues. In conclusion molecular analyses from PET can be greatly improved by using a non-crosslinking fixative and storage at lower temperatures such as 4°C, which should be considered in prospective clinical studies.

Occasionally, tissue samples cannot be processed completely and are stored under varying conditions for extended periods. This is particularly beneficial in interinstitutional studies where a given research setting may lack the expertise or infrastructure for sample processing, imaging and data analysis. Currently, there is limited literature available on the controlled storage of biological tissues in primary fixatives for fluorescence and electron microscopy. In this contribution, we mimicked various tissue storage scenarios by taking different fixation conditions, storage temperatures and storage durations into account. Rat liver tissue was used for its well-known diversity of cellular ultrastructure and microscopy analysis. Fluorescent labelling of actin, DNA and lipids were employed in conjunction with high-resolution electron microscopy imaging. Herein, we tested three different fixative solutions (1.5% glutaraldehyde, 0.4% glutaraldehyde and 4% formaldehyde and 4% formaldehyde) and stored samples for 1–28 days at room temperature and refrigerator temperature. We found that liver tissue can be stored for up to 2 weeks in a 0.4% glutaraldehyde + 4% formaldehyde fixative solution, while still enabling reliable fluorescent labelling and ultrastructural studies. Ultrastructural integrity was eminent up to 1 month using either glutaraldehyde or formaldehyde fixation protocols. When liver tissue is fixed with a mixture of 0.4% glutaraldehyde and 4% formaldehyde and stored at 4 °C, it retains its capacity for electron microscopy analysis for several years, but loses its capacity for reliable fluorescent labelling studies. In conclusion, we demonstrated that liver tissue can be stored for extended periods enabling profound structure–function analysis across length scales.

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.

Understanding the interaction between ligands and membrane proteins is important for drug design and optimization. Although investigation using live cells is desirable, it is not feasible in some circumstances and cell fixation is performed to reduce cell motion and degradation. This study compared the effects of five fixatives, i.e., formaldehyde vapor (FV), paraformaldehyde (PFA), acetone, methanol, and ethanol, on kinetic measurements via the LigandTracer method. We found that all five fixatives exerted insignificant effects on lectin-glycan interaction. However, antibody-receptor interaction is markedly perturbed by coagulant fixatives. The acetone fixation changed the binding of the anti-human epidermal growth factor receptor 2 (HER2) antibody to HER2 on the cell membrane from a 1:2 to a 1:1 binding model, while methanol and ethanol abolished the antibody binding possibly by removal of the HER2 receptors on the cell membrane. The capability of binding was retained when methanol fixation was performed at lower temperatures, albeit with a binding model of 1:1 instead. Moreover, whereas cell morphology does not exert a substantial impact on lectin-glycan interaction, it can indeed modify the binding model of antibody-receptor interaction. Our results provided insights into the selection of fixatives for cell-based kinetic studies.

Demonstration of glycogen can be done in different lesions and is considered diagnostically significant, mainly in some tumors. Glycogen staining is affected by the type of fixative, the temperature of fixation, and the staining technique.Grocott’s methenamine (hexamine) silver technique quality was assessed after four different types of fixatives at two different temperatures [Bouin’s solution, 10% neutral buffered formalin (NBF), 80% alcohol, and Rossman’s solution at room temperature (RT) and 4 °C, for 24 h]. These variables were studied to optimize this technique for glycogen demonstration. Archived paraffin blocks were used in this study. They were prepared from one rabbit’s liver, and 32 paraffin sections were prepared and stained with Grocott’s methenamine (hexamine) silver technique. Eighty percent alcohol provided the highest staining quality scores at both RT and 4 °C in comparison with the other fixatives. We concluded that 80% alcohol at 4 °C seems to be the fixative of choice for glycogen with the Grocott’s methenamine (hexamine) silver technique at the level of this study.

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15 ) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15 . We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics. Programmed cell death or apoptosis is an essential biological process that is impaired in some diseases and can be used to assess the effectiveness of drugs. Here the authors design Apo-15 as a fluorogenic peptide for the detection and real-time imaging of apoptotic cells.

Tissue preservation is a minimal requirement for the success of plant RNA and protein expression studies. The standard of snap-freezing in liquid nitrogen is not always practical or possible. RNAlater, a concentrated solution of ammonium and cesium sulfates, has become a standard preservative in the absence of liquid nitrogen. Here, we demonstrate the effectiveness of RNAlater in preserving both RNA and proteins in Arabidopsis thaliana tissues for use in RNAseq and LC-MS/MS analysis of proteins. While successful in preserving plant material, a transcriptomic and proteomic response is evident. Specifically, 5770 gene transcripts, 84 soluble proteins, and 120 membrane-bound proteins were found to be differentially expressed at a log-fold change of ±1 (P ≤ 0.05). This response is mirrored in the abundance of post-translational modifications, with 23 of the 108 (21.3%) phosphorylated proteins showing altered abundance at a log-fold change of ±1 (P ≤ 0.05). While RNAlater is effective in preserving biological information, our findings warrant caution in its use for transcriptomic and proteomic experiments.

The formation of neutrophil extracellular traps (NETs) is known as an important part of the innate immune response. Still, some mechanisms regarding their formation and role during a disease are not completely understood yet. To visualize NETs by immunofluorescence microscopy, a chemical fixation is required. Therefore, this study focused on the effect of chemical fixatives on immunofluorescence staining of selected neutrophil and NET-markers, including myeloperoxidase (MPO), DNA/histone-1-complexes and citrullinated histone H3 (H3cit). Neutrophils isolated from fresh human blood were stimulated with phorbol-12-myristate 13-acetate (PMA) to induce NETs and fixed with paraformaldehyde (PFA, 4%), glutardialdehyde (GA, 5%) or methanol (MeOH, 100%) using different incubation times depending on the used fixative. We found that different fixation times with PFA had no effect on the staining intensity of MPO or DNA/histone-1-complex antibodies. For the staining of H3cit, fixation with PFA for 24 h decreased the signal intensity whereas 30 min fixation time had no effect. In contrast, glutardialdehyde induced a high amount of autofluorescence, and the fixation with 100% MeOH resulted in visible cellular damage. Therefore, we recommend 15–30 min PFA fixation for the respective stainings. Our results provide a solid basis for future experiments to study neutrophil activation and NET-formation.