Feeder cells treated with ethanol can be used to maintain self-renewal and pluripotency of human pluripotent stem cells.

Feeder cells play an important role in the culture of human pluripotent stem cells (hPSCs) in vitro. Previously, we used methanol as a fixative to prepare feeder cells for the cultivation of pluripotent stem cells (PSCs), and this method could maintain the self-renewal and pluripotency of PSCs. However, methanol is toxic, and so here we examined whether ethanol could be used to prepare feeder cells as a fixative for hPSC culturing. Primed, naïve, and extended human embryonic stem cells and induced pluripotent stem cells can maintain self-renewal and undifferentiated potential on feeder cells treated with ethanol for an extended period. RNA sequencing analysis showed that the expression of collagen-related genes in hPSCs cultured on feeder cells treated with ethanol was significantly lower as compared with hPSCs cultured on feeder cells treated with mitomycin C. Therefore, we speculate that the signaling pathway mediated by collagen-related genes may, at least in part, contribute to the maintenance of self-renewal and pluripotency of PSCs induced by feeder cells treated with chemicals.

Protocol to detect smooth muscle actin-alpha and measure oxidative damage in neonatal mouse intestine.

This protocol describes how to characterize α-Smooth muscle actin (αSMA) spatiotemporal expression during mouse small intestinal development. Specific tissue fixation preserves αSMA arrangement in low αSMA expressing cells that are conventionally undetectable under αSMA immunofluorescent stain due to inappropriate fixative-caused artificial actin depolymerization. Parallel analysis of αSMA carbonylation allows estimation of oxidative damage in gut muscular lineage. This approach improves the molecular specificity offered by commercialized kits that estimate total protein carbonyl level in cell lysates without protein specificity. For complete details on the use and execution of this protocol, please refer to Hu et al. (2021).

Glyoxal Fixation as an Alternative for Zebrafish Embryo Immunostaining.

Immunohistochemistry has been widely used as a robust technique to determine the cellular and subcellular localization of proteins. This information ultimately helps to understand the function of these proteins and how biological processes are regulated. Antibodies applicable for labeling in zebrafish are limited, making immuno-staining challenging. Recently glyoxal fixation was rediscovered in tissue culture, mouse, rat, and Drosophila, expanding the list of effective antibodies for these species. Here, we compare a protocol for zebrafish staining using glyoxal as a fixative agent with PFA. We demonstrate that glyoxal fixation improves the antigenicity of some epitopes thereby increasing the number of useful antibodies in zebrafish.

Assessment of dynamic bioaccessibility of curcumin encapsulated in milled starch particle stabilized Pickering emulsions using TNO's gastrointestinal model.

Pickering emulsions stabilized by milled starch particles have been developed as a novel food-grade formulation to enhance the bioaccessibility of poorly soluble bioactive compounds (i.e., curcumin) by controlling the digestion of lipids in the human gastrointestinal (GI) tract. The dynamic bioaccessibilities of curcumin with and without encapsulation in the Pickering emulsion were evaluated using the dynamic TNO's gastrointestinal (TIM-1) model. For comparison, their digestion profiles were also studied using the in vitro pH-stat lipolysis model. With the combination of two in vitro models, the effect of the milled starch particle stabilized Pickering emulsions on the bioaccessibility of curcumin was fully revealed. There are large differences between the bioaccessibility values of curcumin samples obtained by these two models. Simulated small intestinal lipolysis in the pH-stat model revealed that the bioaccessibility of curcumin encapsulated in the Pickering emulsion was 27.6%, which was larger than 22.1% for free curcumin suspended in the bulk oil phase. The bioaccessibility of curcumin was 50.7% in the emulsion system and 7.8% in the bulk oil when using the TIM-1 model, which simulated the digestion conditions of the entire human GI tract. The digestion mechanism of the milled starch particle stabilized Pickering emulsions in the upper GI tract was well elucidated by the TIM-1 model. The gradual release and improved dissolution profile of the milled starch particle stabilized Pickering emulsions highlighted their potential as delivery systems for lipophilic bioactive compounds.

DNA metabarcoding from sample fixative as a quick and voucher-preserving biodiversity assessment method 1.

Metabarcoding is a powerful, increasingly popular tool for biodiversity assessment, but it still suffers from some drawbacks (specimen destruction, separation, and size sorting). In the present study, we tested a non-destructive protocol that excludes any sample sorting, where the ethanol used for sample preserving is filtered and DNA is extracted from the filter for subsequent DNA metabarcoding. When tested on macroinvertebrate mock communities, the method was widely successful but was unable to reliably detect mollusc taxa. Three different protocols (no treatment, shaking, and freezing) were successfully applied to increase DNA release to the fixative. The protocols resulted in similar success in taxa detection (6.8-7 taxa) but differences in read numbers assigned to taxa of interest (33.8%-93.7%). In comparison to conventional bulk sample metabarcoding of environmental samples, taxa with pronounced exoskeleton and small-bodied taxa were especially underrepresented in ethanol samples. For EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa, which are important for determining stream ecological status, the methods detected 46 OTUs in common, with only 4 unique to the ethanol samples and 10 to the bulk samples. These results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. However, for a comprehensive assessment on total invertebrate biodiversity, the method may not be sufficient, and conventional bulk sample metabarcoding should be applied.

Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation.

To understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes with long acquisition times and high phototoxicity. Therefore, physiological live cell imaging is often unfeasible and chemical fixation is employed. Yet, fixation methods have not been rigorously investigated, in terms of pattern preservation, at the resolution at which cells can now be imaged. A key parameter for this is the time required until fixation is complete. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Other small aldehydes, glyoxal and acrolein, did not perform better. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from cells. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely absent with supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

The effects of chemical fixation on the cellular nanostructure.

Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo-fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever-finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label-free live cell spectroscopic-imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo-fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods.

Preparing Fission Yeast for Electron Microscopy.

Freezing samples while simultaneously subjecting them to a rapid increase in pressure, which inhibits ice crystal formation, is a reliable method for cryofixing fission yeast. The procedure consists simply of harvesting cells and loading them into a high-pressure freezer (HPF), and then operating the device. If equipment for high-pressure freezing is not available, fission yeast can be frozen by plunging a monolayer of cells into a liquid cryogen, usually ethane or propane. Unlike the HPF, where relatively large volumes of cells can be frozen in a single run, plunge freezing requires cells to be dispersed in a layer <20 µm thick. Unless frozen cells are to be imaged in the vitreous state, they must be fixed, dehydrated, and embedded for subsequent study by transmission electron microscopy; warming frozen cells without fixation badly damages cell structure. Fixation is best accomplished by freeze-substitution, a process in which frozen water is removed from samples by a water-miscible solvent that is liquid at a temperature low enough to prevent the cellular water from recrystallizing. Low concentrations of chemical fixatives and stains are generally added to this solvent such that they permeate the cells as the water is replaced. The activity of these additives is quite limited at the low temperatures required for minimizing ice crystal formation, but they are in the right place to react effectively as the cells warm up. Step-by-step protocols for HPF, plunge freezing, and freeze-substitution are provided here.

Immunofluorescence Microscopy of Schizosaccharomyces pombe Using Chemical Fixation.

Establishing the subcellular distribution of molecules of interest and the dynamics of their spatial control underpins all areas of cell and developmental biology. Although the ability to monitor the distribution of fluorescent fusion proteins has revolutionized cell and developmental biology, indirect immunofluorescence microscopy of fixed samples remains an essential complement to this approach. Immunofluorescence is often a more appropriate approach for the study of subcellular architecture. It avoids potential artifacts caused by studying fusion proteins, which might show altered function under stressful imaging conditions. Furthermore, the quantitative analysis of multiple cells in an unperturbed population by immunofluorescence invariably provides a more accurate assessment of the spatial and temporal control of a particular process than does the analysis of individual cells that is the hallmark of live-cell imaging. Parallel studies of living and fixed cells often provide complementary data sets, both of which can be considered necessary for a comprehensive understanding of molecular function. This protocol provides a method for the visualization of the Schizosaccharomyces pombe microtubule cytoskeleton by indirect immunofluorescence microscopy following chemical fixation with formaldehyde and glutaraldehyde. It includes discussion of common modifications used to monitor the distribution of other fission yeast antigens and forms a basis from which to develop protocols to localize new molecules of interest.

Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation.

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.

Pre-fixation of virulent Mycobacterium tuberculosis with glutaraldehyde preserves exquisite ultrastructure on transmission electron microscopy through cryofixation and freeze-substitution with osmium-acetone at ultralow temperature.

Sample preparations for transmission electron microscopy of virulent Mycobacterium tuberculosis are usually performed with chemical fixation using glutaraldehyde (GA) in a biosafety area followed by post-fixation with aqueous osmium tetroxide (OT) in a conventional laboratory outside the biosafety area. Freeze-substitution with osmium-acetone (OA) at ultralow temperature (-85°C) has been shown to provide high quality final images and preserves cellular structures intact. However, some preparation procedures for freeze-substitution often require large fixed devices for freezing in a special laboratory. We have reported a novel freeze-substitution preparation method that can be performed using a portable device in a biosafety cabinet at biosafety level (BSL) 3 areas. Here, as a next step, we examined whether images obtained from rapid freeze-substitution (RFS) after fixation with glutaraldehyde (GA>RFS) are of comparable quality to those obtained using standard RFS. GA>RFS provided excellent preservation of mycobacterial cell ultrastructure, including visualization of cytoplasmic ribosomes, DNA fibers, and the outer membrane. The average number of ribosomes per cubic micrometer counted on RFS and GA>RFS was not significantly different (6987.8±2181.0 and 6888.9±1799.3, respectively). These values were higher, but not significantly so, than those obtained using conventional chemical fixation (5018.7±2511.3). This procedure may be useful for RFS preparation of unculturable mycobacteria strains or virulent strains isolated in laboratories that cannot perform RFS.

Feasibility of high pressure freezing with freeze substitution after long-term storage in chemical fixatives.

Fixation of biological samples is an important process especially related to histological and ultrastructural studies. Chemical fixation was the primary method of fixing tissue for transmission electron microscopy for many years, as it provides adequate preservation of the morphology of cells and organelles. High pressure freezing (HPF) and freeze substitution (FS) is a newer alternative method that rapidly freezes non-cryoprotected samples that are then slowly heated in the FS medium, allowing penetration of the tissue to insure adequate fixation. This study addresses several issues related to tissue preservation for electron microscopy. Using mice liver tissue as model the difference between samples fixed chemically or with HPF immediately after excision, or stored before chemical or HPF fixation were tested with specific focus on the nuclear membrane. Findings are that immediate HPF is the method of choice compared to chemical fixation. Of the chemical fixatives, immediate fixation with 2.5% glutaraldehyde (GA)/formaldehyde (FA) is the best in preserving membrane morphology, 2.5% GA can be used as alternative for stored and then chemically processed samples, with 10% formalin being suitable as a storage medium only if followed by HPF fixation. Overall, storage leads to lower ultrastructural preservation, but HPF with FS can minimize these artifacts relative to other processing protocols.

Development of a continuous bioconversion system using a thermophilic whole-cell biocatalyst.

The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.

Immunization of mice with formalin-inactivated spores from avirulent Bacillus cereus strains provides significant protection from challenge with Bacillus anthracis Ames.

Bacillus anthracis spores are the infectious form of the organism for humans and animals. However, the approved human vaccine in the United States is derived from a vegetative culture filtrate of a toxigenic, nonencapsulated B. anthracis strain that primarily contains protective antigen (PA). Immunization of mice with purified spore proteins and formalin-inactivated spores (FIS) from a nonencapsulated, nontoxigenic B. anthracis strain confers protection against B. anthracis challenge when PA is also administered. To investigate the capacity of the spore particle to act as a vaccine without PA, we immunized mice subcutaneously with FIS from nontoxigenic, nonencapsulated B. cereus strain G9241 pBCXO1(-)/pBC210(-) (dcG9241), dcG9241 ΔbclA, or 569-UM20 or with exosporium isolated from dcG9241. FIS vaccination provided significant protection of mice from intraperitoneal or intranasal challenge with spores of the virulent B. anthracis Ames or Ames ΔbclA strain. Immunization with dcG9241 ΔbclA FIS, which are devoid of the immunodominant spore protein BclA, provided greater protection from challenge with either Ames strain than did immunization with FIS from BclA-producing strains. In addition, we used prechallenge immune antisera to probe a panel of recombinant B. anthracis Sterne spore proteins to identify novel immunogenic vaccine candidates. The antisera were variably reactive with BclA and with 10 other proteins, four of which were previously tested as vaccine candidates. Overall our data show that immunization with FIS from nontoxigenic, nonencapsulated B. cereus strains provides moderate to high levels of protection of mice from B. anthracis Ames challenge and that neither PA nor BclA is required for this protection.

Formaldehyde fixation of Drosophila testes.

This protocol describes formaldehyde fixation of Drosophila testes. The procedure preserves chromosome morphology very well, allowing a clear visualization of the chromosome condensation-decondensation cycle. It also results in excellent preservation of microtubules for immunostaining. The main disadvantage of this fixation technique is its poor preservation of cell morphology, as viewed by phase-contrast optics. In addition, because it involves a rather hard squashing of formaldehyde-fixed testes, it results in meiotic figures which are flatter and larger than those obtained using methanol-acetone or paraformaldehyde fixation protocols.

Formaldehyde fixation of Drosophila tissues onto slides for whole-mount FISH.

This protocol describes the formaldehyde fixation of Drosophila tissues other than egg chambers. It provides a general approach to fixing tissue or cells onto microscope slides in a manner compatible with whole-mount fluorescent in situ hybridization (FISH) analysis. The procedure includes a simple formaldehyde fixation in buffer, followed by postfixation in cold ethanol. Postfixation of the tissue in methanol or ethanol can markedly improve permeability. In some instances, it may be preferable to fix tissue onto coverslips, because microscope optics are usually designed to optimize imaging immediately adjacent to the coverslip. On the other hand, coverslips are much more fragile and harder to manipulate.

Transmission electron microscopy of thin sections of Drosophila: conventional chemical fixation of embryos using trialdehyde.

High-pressure freezing (HPF) followed by freeze-substitution is a valuable method for specimen preservation for transmission electron microscopy (TEM) in Drosophila. However, not all projects require this level of precision. In addition, some tissues are too large to fit into the HPF specimen carriers, and some fly tissues such as eyes and ovaries do not freeze well. This protocol describes a trialdehyde fixation procedure for embryos, to be used in situations where optimal preservation is not required or when HPF is not an option. Because the vitelline membrane is impermeable to aqueous solvents, it is necessary to either mechanically disrupt it or render it permeable by treatment with organic solvents. Good ultrastructural preservation has been achieved by puncturing embryos immersed in fixative with extremely sharp tungsten needles, as described here.

Paraformaldehyde fixation of Drosophila testes.

This protocol describes paraformaldehyde fixation of Drosophila testes. The procedure preserves F-actin-containing structures because it does not involve the use of methanol. Thus, it is particularly suitable for contractile ring and fusome visualization. However, it does not preserve cell morphology and microtubules as efficiently as methanol-acetone fixation and does not permit clear detection of the Y loops.

F-actin staining of Drosophila testes.

Preparations of Drosophila testes fixed with paraformaldehyde can be stained for F-actin according to the protocol described here. This staining procedure is particularly suitable for staining the male fusome and the cytokinetic contractile ring.

Improved polyacrylamide-based artificial sputum with formalin-fixed tubercle bacilli for training of tuberculosis microscopists.

Sputum smear microscopy is an easy, inexpensive, and rapid method for detecting tubercle bacilli when there are more than 10,000 bacilli/ml in the original sputum. Furthermore, because the microscopic method provides not only quantitative, but also qualitative information, such as the shape of bacilli, it has remained significant. We have previously developed and reported panel test slides made from polyacrylamide-based artificial sputum (PBAS) mixed with both cultured THP-1 cells and nonpathogenic mycobacteria. In this paper, we report an improved preparation method for PBAS for panel test slides that provides a simplified method and enhanced availability with high consistency in each grade and in which only negative PBAS is prepared from polyacrylamide and cultured THP-1 cells and mixed with graded formalin-fixed Mycobacterium tuberculosis solution (FFTBS) containing oral flora and Pseudomonas aeruginosa on the slides. In the smears prepared using this improved method, the numbers (average ± standard deviation [SD]) of acid-fast bacilli (AFB) in 300 fields (2- by 3-cm smear) in eight smears of each grade ranged from 5 to 9 (6.4 ± 1.4), from 59 to 88 (74.6 ± 10.0), from 503 to 912 (705.0 ± 145.7), and from 1,819 to 3,256 (2133.3 ± 478.0) in ±, +, ++, and +++ smears, respectively. In addition, this preparation method provided high similarity to the microscopic appearance of bacilli and background seen in the actual patient sputum, with high feasibility. These results revealed that our new PBAS had high authenticity in the appearance and consistency in each grade, which could make it valuable as a reliable artificial sputum for the training of microscopists.