Fixation Step Research Articles

Fluorescence Approaches to Image and Quantify the Demarcation Membrane System in Living Megakaryocytes.

The demarcation membrane system (DMS) develops to provide additional surface membrane for the process of platelet production. The DMS is an invagination of the plasma membrane that can extend throughout the extranuclear volume of mature megakaryocytes and its lumen is continuous with the extracellular solution. DMS ultrastructure in fixed samples has been extensively studied using transmission electron microscopy (TEM) and more recently with focused ion beam scanning EM. In addition, whole cell patch clamp membrane capacitance provides a direct measurement of DMS content in living megakaryocytes. However, fluorescence methods to image and quantify the DMS in living megakaryocytes provide several advantages. For example, confocal fluorescence microscopy is easier to use compared to EM or electrophysiological methods and the required equipment is more readily available. In addition, use of living cells avoids artifacts known to occur during the fixation, dehydration, or embedding steps used to prepare EM samples. Here we describe the use of styryl dyes such as FM 1-43 or di-8-ANEPPS and impermeant fluorescent indicators of the extracellular space as simple approaches for imaging and quantification of the DMS.

A simple and fast method for fixation of cultured cell lines that preserves cellular structures containing gamma-tubulin

When using fluorescence microscope techniques to study cells, it is essential that the cell structure and contents are preserved after preparation of the samples, and that the preparation method employed does not create artefacts that can be perceived as cellular structure/components. ?-Tubulin forms filaments that in some cases are immunostained with an anti-?-tubulin antibody, but this immunostaining is not reproducible [1]. In addition, the C terminal region of ?-tubulin (green fluorescence protein tagged [GFP]-?-tubulin334––449) forms cytosolic GFP-labeled structures, which can easily be imaged in live cells but are not preserved in fixed cells [1,2]. The purpose of this study was to identify a fixation technique that preserves cellular constituents containing ?-tubulin.•This protocol describes a method that preserves ?-tubulin-containing structures in fixed cells.•The technique entails two-step fixation. A pre-fixation step using paraformaldehyde is followed by a final fixation and permeabilization step performed at -80?°C.•In comparison with other methodology for fixation [3–5], the technique presented here uses a short pre-fixation step with a mixture of paraformaldehyde and sucrose followed by a short fixation/permeabilization step with a mixture of methanol and acetone at -80?°C.

Counterion Dye Staining of Proteins in One- and Two-Dimensional Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Tryptic Gel Digestion of Stained Protein for Mass Spectrometry.

A fast and matrix-assisted laser desorption/ionization-mass spectrometry compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon and ethyl violet to form an ion-pair complex. The protocol including fixing, staining, and quick washing steps can be completed in 1-1.5h depending upon gel thickness. It has the sensitivity comparable to the colloidal Coomassie Brilliant Blue G stain using phosphoric acid as a component of staining solution (4-8ng). The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from mass spectrometry. Considering the speed, sensitivity, and compatibility with mass spectrometry, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches.

A comprehensive molecular and morphological study of the effects of space flight on human capillary endothelial cells: sample quality assessment and preliminary results.

ESA (ESA ILSRA-2009-1026); ASI (contract no. 5681); Regione Toscana (POR FSE 2007-2013-FORTEC); Kayser Italia; Lions Club International, District 108LA, Toscana, Italy.

An autofluorescence-based method for the isolation of highly purified ventricular cardiomyocytes.

The aim of our study was to set up a simple and reliable isolation method of living ventricular cardiomyocytes (vCMs) for molecular and biological studies. A standard technique for the retrograde perfusion of an enzymatic solution was used to isolate cardiac cells from adult mouse heart. Fluorescence-activated cell sorting (FACS) on adult murine cardiac ventricle cells was performed, comparing the intrinsic autofluorescence in the FITC channel and the forward scatter (FSC) parameter in order to isolate highly fluorescent cells. The expression of cell-specific mRNAs was assessed with real-time PCR in cells sorted on the basis of their FITC and FSC characteristics. We identified two distinct subpopulations of cells harvested after retrograde perfusion of wild-type heart: FITChigh/FSCdim and FITCdim/FSChigh. Immunophenotyping and mRNA analysis (qPCR and RNA sequencing) revealed that only FITChigh/FSCdim cells were highly enriched in CM markers. Genes with high expression in endothelial cells and fibroblasts were enriched in the FITCdim/FSChigh subpopulation. With the use of tdTomatofl/fl-α-myosin heavy chain MerCreMer+/-mouse heart, we found that tdTomato-positive vCMs were present in the FITChigh/FSCdim region but were only rare in the FITCdim/FSChigh fraction. We have developed a simple and reliable method for the isolation of highly purified vCMs from the adult murine myocardium, avoiding fixation and permeabilization steps. These isolated vCMs can be used in particular for detailed molecular studies, avoiding contamination with other myocardial cell types.

Understanding of Electrochemical Mechanisms for CO2 Capture and Conversion into Hydrocarbon Fuels in Transition-Metal Carbides (MXenes).

Two-dimensional (2D) transition-metal (groups IV, V, VI) carbides (MXenes) with formulas M3C2 have been investigated as CO2 conversion catalysts with well-resolved density functional theory calculations. While MXenes from the group IV to VI series have demonstrated an active behavior for the capture of CO2, the Cr3C2 and Mo3C2 MXenes exhibit the most promising CO2 to CH4 selective conversion capabilities. Our results predicted the formation of OCHO• and HOCO• radical species in the early hydrogenation steps through spontaneous reactions. This provides atomic level insights into the computer-aided screening for high-performance catalysts and the understanding of electrochemical mechanisms for CO2 reduction to energy-rich hydrocarbon fuels, which is of fundamental significance to elucidate the elementary steps for CO2 fixation.

Application of laboratory and digital techniques for visual enhancement during the ultrastructural assessment of cilia

ABSTRACTRoutine diagnostic electron microscopy of primary ciliary dyskinesia (PCD) is based on the findings of ultrastructural defects of axonemal components. Assessment of the typical abnormalities can be enhanced by improving the sample preservation status using tannic acid (TA) as additive in the biopsy fixation or processing steps. Another option is the implementation of computer-assisted image analysis tools. Advancements in high-resolution 3D visualization of the axonemal structure have been noted, with great potential for the future diagnosis of inherited cilia disorders.

Permeabilization-Tolerant Plasma Membrane Imaging Reagent Based on Amine-Rich Glycol Chitosan Derivatives.

Immunofluorescence staining is a crucial tool for studying the structure and behavior of intracellular proteins and organelles. During the staining process, the permeabilization treatment is usually required to enhance the penetration of a fluorescent antibody into the cells. However, since most of the membrane imaging dyes as well as the membrane lipids will detach from the cell surface after permeabilization, membrane labeling using these dyes is not compatible with immunofluorescence staining. Herein, by linking cholesterol-polyethylene glycol (PEG-Chol) and fluorescein isothiocyanate (FITC) with the amine-rich glycol chitosan (GC), we prepared a multifunctional polymeric construct, GC-PEG Chol-FITC, and realized permeabilization-tolerant plasma membrane imaging. Owing to the presence of abundant amine groups in the labeling reagent and the membrane proteins/lipids, the addition of paraformaldehyde in the fixation step induces the amine-cross-linking between the labeling reagents and the membrane proteins/lipids, thus preventing the detachment of fluorophores from the cell surface after permeabilization. Besides, the large molecular weight effect of the imaging reagent may also account for its antipermeabilization property. Furthermore, by combining immunofluorescence staining with the plasma membrane labeling by GC-PEG Chol-FITC, we simultaneously imaged the plasma membrane and cytoskeletons, and clearly observed metaphase cells and binucleated cells. The concept of using amine-rich polymeric dyes for plasma membrane imaging will inspire the development of more permeabilization-resistant membrane labeling dyes with better performance, which can realize simultaneous membrane and intracellular protein imaging and facilitate the future studies of membrane-intracellular protein interactions.

Unraveling RubisCO Form I and Form II Regulation in an Uncultured Organism from a Deep-Sea Hydrothermal Vent via Metagenomic and Mutagenesis Studies.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first major step of carbon fixation in the Calvin-Benson-Bassham (CBB) cycle. This autotrophic CO2 fixation cycle accounts for almost all the assimilated carbon on Earth. Due to the primary role that RubisCO plays in autotrophic carbon fixation, it is important to understand how its gene expression is regulated and the enzyme is activated. Since the majority of all microorganisms are currently not culturable, we used a metagenomic approach to identify genes and enzymes associated with RubisCO expression. The investigated metagenomic DNA fragment originates from the deep-sea hydrothermal vent field Nibelungen at 8°18′ S along the Mid-Atlantic Ridge. It is 13,046 bp and resembles genes from Thiomicrospira crunogena. The fragment encodes nine open reading frames (ORFs) which include two types of RubisCO, form I (CbbL/S) and form II (CbbM), two LysR transcriptional regulators (LysR1 and LysR2), two von Willebrand factor type A (CbbO-m and CbbO-1), and two AAA+ ATPases (CbbQ-m and CbbQ-1), expected to function as RubisCO activating enzymes. In silico analyses uncovered several putative LysR binding sites and promoter structures. Functions of some of these DNA motifs were experimentally confirmed. For example, according to mobility shift assays LysR1’s binding ability to the intergenic region of lysR1 and cbbL appears to be intensified when CbbL or LysR2 are present. Binding of LysR2 upstream of cbbM appears to be intensified if CbbM is present. Our study suggests that CbbQ-m and CbbO-m activate CbbL and that LysR1 and LysR2 proteins promote CbbQ-m/CbbO-m expression. CbbO-1 seems to activate CbbM and CbbM itself appears to contribute to intensifying LysR’s binding ability and thus its own transcriptional regulation. CbbM furthermore appears to impair cbbL expression. A model summarizes the findings and predicts putative interactions of the different proteins influencing RubisCO gene regulation and expression.

A rapid and effective method for observation of suberized cell layers in potato tuber skin

The suberization of surface cells is important for potato tubers for water and mineral metabolisms, and disease resistance during growth and development. Observation of structural changes of suberized cell layers can provide the most constructive logical evidence for related research. A quick and effective method to observe suberized cell layer in potato tuber skin is reported in this paper. This improved method removed the common fixation steps and reduced staining time to only 5–10min. The process to analyze a tuber sample takes less than 30min. Various types of potato tubers, including mature and immature tubers, PCS (potato common scab) pathogen infected tuber and microtubers were used in this study. Qualified observations were obtained and are presented. The method provides an alternative for research related to tuber skin studies, such as potato tuber disease development, maturation, and storage quality.

COMPARAÇÃO ENTRE AS TÉCNICAS ANATÔMICAS DE FORMOLIZAÇÃO, GLICERINAÇÃO E PREENCHIMENTO/CORROSÃO NO SISTEMA RESPIRATÓRIO DE CÃES

The objective of the techniques of conservation of anatomical pieces is to preserve the morphology, consistency, color and flexibility of the same. Among the techniques currently used are formaldehyde, glycerin and fill/corrosion, which were compared in the present study. Four pieces were used in the evaluation of each technique. For formalinization, the lungs were washed, dissected and fixed in 10% formalin. For the glycerination technique, the organs were fixed in 10% formaldehyde, washed for 48 hours, and then dried in the shade. Sequentially, the organs were submerged in hydrogen peroxide 10 and again dried in the shade. Then, they remained submerged in absolute alcohol for two months, were removed, dried naturally, and submerged in glycerin for 30 days. For the filling/corrosion technique the lungs were washed, then submerged in 125 Colloquium Agrariae, vol. 13, n. Especial 2, Jan–Jun, 2017, p. 124-128. ISSN: 1809-8215. DOI: 10.5747/ca.2017.v13.nesp2.000217 alcohol for 24 hours. The trachea was cannulated for the fixation step, through the canula a continuous air stream containing alcohol and formalin mixture was introduced into the bronchial tree. Subsequently, the acrylic was inserted, the pieces were placed in the water for 24 hours, and soon the corrosion was carried out with hydrochloric acid. The glycerolation technique proved to be more advantageous when compared to formaldehyde, since it presents a low toxicity and irritation index, which facilitated the management and study, besides requiring a low degree of maintenance. However, for the visualization of the bronchial tree, the filling/corrosion technique proved to be superior to the others, but this technique demands greater care during its handling and storage.

Iron catalysis at the origin of life.

Iron-sulphur proteins are ancient and drive fundamental processes in cells, notably electron transfer and CO2 fixation. Iron-sulphur minerals with equivalent structures could have played a key role in the origin of life. However, the 'iron-sulphur world' hypothesis has had a mixed reception, with questions raised especially about the feasibility of a pyrites-pulled reverse Krebs cycle. Phylogenetics suggests that the earliest cells drove carbon and energy metabolism via the acetyl CoA pathway, which is also replete in Fe(Ni)S proteins. Deep differences between bacteria and archaea in this pathway obscure the ancestral state. These differences make sense if early cells depended on natural proton gradients in alkaline hydrothermal vents. If so, the acetyl CoA pathway diverged with the origins of active ion pumping, and ancestral CO2 fixation might have been equivalent to methanogens, which depend on a membrane-bound NiFe hydrogenase, energy converting hydrogenase. This uses the proton-motive force to reduce ferredoxin, thence CO2 . The mechanism suggests that pH could modulate reduction potential at the active site of the enzyme, facilitating the difficult reduction of CO2 by H2 . This mechanism could be generalised under abiotic conditions so that steep pH differences across semi-conducting Fe(Ni)S barriers drives not just the first steps of CO2 fixation to C1 and C2 organics such as CO, CH3 SH and CH3 COSH, but a series of similar carbonylation and hydrogenation reactions to form longer chain carboxylic acids such as pyruvate, oxaloacetate and α-ketoglutarate, as in the incomplete reverse Krebs cycle found in methanogens. We suggest that the closure of a complete reverse Krebs cycle, by regenerating acetyl CoA directly, displaced the acetyl CoA pathway from many modern groups. A later reliance on acetyl CoA and ATP eliminated the need for the proton-motive force to drive most steps of the reverse Krebs cycle. © 2017 IUBMB Life, 69(6):373-381, 2017.

Engineering chloroplasts to improve Rubisco catalysis: prospects for translating improvements into food and fiber crops.

494 I. 495 II. 496 III. 496 IV. 499 V. 499 VI. 501 VII. 501 VIII. 502 IX. 505 X. 506 507 References 507 SUMMARY: The uncertainty of future climate change is placing pressure on cropping systems to continue to provide stable increases in productive yields. To mitigate future climates and the increasing threats against global food security, new solutions to manipulate photosynthesis are required. This review explores the current efforts available to improve carbon assimilation within plant chloroplasts by engineering Rubisco, which catalyzes the rate-limiting step of CO2 fixation. Fixation of CO2 and subsequent cycling of 3-phosphoglycerate through the Calvin cycle provides the necessary carbohydrate building blocks for maintaining plant growth and yield, but has to compete with Rubisco oxygenation, which results in photorespiration that is energetically wasteful for plants. Engineering improvements in Rubisco is a complex challenge and requires an understanding of chloroplast gene regulatory pathways, and the intricate nature of Rubisco catalysis and biogenesis, to transplant more efficient forms of Rubisco into crops. In recent times, major advances in Rubisco engineering have been achieved through improvement of our knowledge of Rubisco synthesis and assembly, and identifying amino acid catalytic switches in the L-subunit responsible for improvements in catalysis. Improving the capacity of CO2 fixation in crops such as rice will require further advances in chloroplast bioengineering and Rubisco biogenesis.

Analyzing ERK Signal Dynamics During Zebrafish Somitogenesis.

During vertebrate development, Erk is activated and regulates multiple cellular processes such as cell growth, differentiation, migration, and adhesion in a spatiotemporal manner. Whole-mount immunohistochemistry using antibodies against diphosphorylated Erk (p-Erk; active form of Erk) is a very useful method for understanding the spatial and temporal patterns of Erk activity during embryonic development. However, the fixation step of this method stops embryo development at a certain time point, making it very difficult to observe and interpret Erk activity dynamics. In this chapter, we describe a strategy that combines immunohistochemistry and quantitative analyses of multiple fixed embryos to reconstruct Erk activity dynamics during zebrafish somitogenesis.

Recent advances in CO2 uptake and fixation mechanism of cyanobacteria and microalgae

ABSTRACTAquatic photosynthetic microorganisms, cyanobacteria and microalgae, account for almost half of the world's photosynthesis. They absorb carbon di oxide (CO2) as the major substrate to support photosynthesis, the beginning of energy flow into living organisms and one of the primary processes comprising the global carbon cycle. Among all photosynthetic mechanisms, inorganic carbon transport into ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is one of the major limiting steps in photosynthetic carbon fixation which involves active transport of HCO3−, CO2 and/or H+, or an energized biochemical mechanism. In fact, a unique system “carbon concentrating mechanism (CCM)” manages the inorganic carbon assimilation, accumulation of CO2 around RuBisCO, and utilization in algal cells. However, the information on mechanism of CO2 uptake and fixation inside the algal cells is limited. In order to make strategies for enhancement of CO2 fixation, understanding of CCM is crucial. Thus, this review provides an overview of advances in CCM research, the comparative state of the art and reports on the CO2 uptake model in cyanobacteria and microalgae. The review also discusses the challenges and future perspectives associated with algal CCM research.

High-contrast X-ray micro-radiography and micro-CT of ex-vivo soft tissue murine organs utilizing ethanol fixation and large area photon-counting detector.

Using dedicated contrast agents high-quality X-ray imaging of soft tissue structures with isotropic micrometre resolution has become feasible. This technique is frequently titled as virtual histology as it allows production of slices of tissue without destroying the sample. The use of contrast agents is, however, often an irreversible time-consuming procedure and despite the non-destructive principle of X-ray imaging, the sample is usually no longer usable for other research methods. In this work we present the application of recently developed large-area photon counting detector for high resolution X-ray micro-radiography and micro-tomography of whole ex-vivo ethanol-preserved mouse organs. The photon counting detectors provide dark-current-free quantum-counting operation enabling acquisition of data with virtually unlimited contrast-to-noise ratio (CNR). Thanks to the very high CNR even ethanol-only preserved soft-tissue samples without addition of any contrast agent can be visualized in great detail. As ethanol preservation is one of the standard steps of tissue fixation for histology, the presented method can open a way for widespread use of micro-CT with all its advantages for routine 3D non-destructive soft-tissue visualisation.

Endoplasmic Reticulum Body-Related Gene Expression in Different Root Zones of Arabidopsis Isolated by Laser-Assisted Microdissection.

Endoplasmic reticulum (ER) bodies are important organelles for root defense. However, little is known regarding the genetic control of their formation in root tissues. In the present study, (L.) Heynh. roots were dissected using laser-assisted microdissection (LAM) with minimal sample preparation (no fixation or embedding steps) and the expression of genes associated with ER body formation and function was assessed by real-time quantitative reverse-transcription polymerase chain reaction (RT-qRT-PCR) in the presence and absence of the defense phytohormone methyl jasmonate (MeJA). Zones of interest were identified in plants overexpressing a fluorescent construct; these being the root cap zone, meristematic zone, elongation zone, and differentiation zone. Given their role in ER body formation, the expression of the genes , , , , and was evaluated in the whole root and in the four dissected root zones using RT-qRT-PCR. Our data show that the expression level of all five genes differs in a root-zone-specific manner in untreated roots. They also reveal that all of them are overexpressed in response to MeJA with the two genes being the most highly overexpressed in the EZ. Finally, the gene, encoding for a transcription factor that regulates the expression of the four other genes, is the first to respond to MeJA, supporting its central role in ER body formation and function in root defense.

Surgical anatomy of medial open-wedge high tibial osteotomy: crucial steps and pitfalls.

To give an overview of the basic knowledge of the functional surgical anatomy of the proximal lower leg and the popliteal region relevant to medial high tibial osteotomy (HTO) as key anatomical structures in spatial relation to the popliteal region and the proximal tibiofibular joint are usually not directly visible and thus escape a direct inspection. The surgical anatomy of the human proximal lower leg and its relevance for HTO are illustrated with a special emphasis on the individual steps of the operation involving creation of the osteotomy planes and plate fixation. The posteriorly located popliteal neurovascular bundle, but also lateral structures such as the peroneal nerve, the head of the fibula and the lateral collateral ligament must be protected from the instruments used for osteotomy. Neither positioning the knee joint in flexion, nor the posterior thin muscle layer of the popliteal muscle offers adequate protection of the popliteal neurovascular bundle when performing the osteotomy. Tactile feedback through a loss-of-resistance when the opposite cortex is perforated is only possible when sawing and drilling is performed in a pounding fashion. Kirschner wires with a proximal thread, therefore, always need to be introduced under fluoroscopic control. Due to anatomy of the tibial head, the tibial slope may increase inadvertently. Enhanced surgical knowledge of anatomical structures that are at a potential risk during the different steps of osteotomy or plate fixation will help to avoid possible injuries. Expert opinion, Level V.

The Roles of Organic Acids in C4 Photosynthesis.

Organic acids are involved in numerous metabolic pathways in all plants. The finding that some plants, known as C4 plants, have four-carbon dicarboxylic acids as the first product of carbon fixation showed these organic acids play essential roles as photosynthetic intermediates. Oxaloacetate (OAA), malate, and aspartate (Asp) are substrates for the C4 acid cycle that underpins the CO2 concentrating mechanism of C4 photosynthesis. In this cycle, OAA is the immediate, short-lived, product of the initial CO2 fixation step in C4 leaf mesophyll cells. The malate and Asp, resulting from the rapid conversion of OAA, are the organic acids delivered to the sites of carbon reduction in the bundle-sheath cells of the leaf, where they are decarboxylated, with the released CO2 used to make carbohydrates. The three-carbon organic acids resulting from the decarboxylation reactions are returned to the mesophyll cells where they are used to regenerate the CO2 acceptor pool. NADP-malic enzyme-type, NAD-malic enzyme-type, and phosphoenolpyruvate carboxykinase-type C4 plants were identified, based on the most abundant decarboxylating enzyme in the leaf tissue. The genes encoding these C4 pathway-associated decarboxylases were co-opted from ancestral C3 plant genes during the evolution of C4 photosynthesis. Malate was recognized as the major organic acid transferred in NADP-malic enzyme-type C4 species, while Asp fills this role in NAD-malic enzyme-type and phosphoenolpyruvate carboxykinase-type plants. However, accumulating evidence indicates that many C4 plants use a combination of organic acids and decarboxylases during CO2 fixation, and the C4-type categories are not rigid. The ability to transfer multiple organic acid species and utilize different decarboxylases has been suggested to give C4 plants advantages in changing and stressful environments, as well as during development, by facilitating the balance of energy between the two cell types involved in the C4 pathway of CO2 assimilation. The results of recent empirical and modeling studies support this suggestion and indicate that a combination of transferred organic acids and decarboxylases is beneficial to C4 plants in different light environments.

Posing for a picture: vesicle immobilization in agarose gel.

Taking a photo typically requires the object of interest to stand still. In science, imaging is potentiated by optical and electron microscopy. However, living and soft matter are not still. Thus, biological preparations for microscopy usually include a fixation step. Similarly, immobilization strategies are required for or substantially facilitate imaging of cells or lipid vesicles, and even more so for acquiring high-quality data via fluorescence-based techniques. Here, we describe a simple yet efficient method to immobilize objects such as lipid vesicles with sizes between 0.1 and 100 μm using agarose gel. We show that while large and giant unilamellar vesicles (LUVs and GUVs) can be caged in the pockets of the gel meshwork, small molecules, proteins and micelles remain free to diffuse through the gel and interact with membranes as in agarose-free solutions, and complex biochemical reactions involving several proteins can proceed in the gel. At the same time, immobilization in agarose has no adverse effect on the GUV size and stability. By applying techniques such as FRAP and FCS, we show that the lateral diffusion of lipids is not affected by the gel. Finally, our immobilization strategy allows capturing high-resolution 3D images of GUVs.