Double cone-unit laparoscopic hepatic resection using indocyanine green negative counterstaining (with video).

A hepatic cone-unit represents an anatomical unit dominated by a smaller Glissonean pedicle. Anatomical resection of a tumor located in an intersegmental plane is challenging, but could be achieved effectively by performing multiple cone-unit resection. We performed double cone-unit laparoscopic resection of hepatocellular carcinoma located on the intersegmental plane between segments 6a, b. The liver parenchyma covering the posterior Glissonean pedicle was divided along Rouviere's sulcus, the Glissonean branches of segments 6a, b were isolated and ligated, and indocyanine green (ICG) negative counterstaining was performed. The hepatic parenchyma was dissected along the demarcation line to identify the right hepatic vein and the double cone-unit resection was then completed with a negative surgical margin. Thus, double cone-unit laparoscopic hepatectomy with ICG negative counterstaining may be a feasible option for tumors located in an intersegmental plane.

A method for rapid and sensitive negative staining of proteins in SDS-PAGE using 2',7'-dichlorofluorescein.

We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS-PAGE by using 2', 7'-dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1-0.2 ng protein could be easily visualized within 7 min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole-zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.

Optimized negative-staining electron microscopy for lipoprotein studies.

BACKGROUND: Negative-staining (NS), a rapid, simple and conventional technique of electron microscopy (EM), has been commonly used to initially study the morphology and structure of proteins for half a century. Certain NS protocols however can cause artifacts, especially for structurally flexible or lipid-related proteins, such as lipoproteins. Lipoproteins were often observed in the form of rouleau as lipoprotein particles appeared to be stacked together by conventional NS protocols. The flexible components of lipoproteins, i.e. lipids and amphipathic apolipoproteins, resulted in the lipoprotein structure being sensitive to the NS sample preparation parameters, such as operational procedures, salt concentrations, and the staining reagents. SCOPE OF REVIEW: The most popular NS protocols that have been used to examine lipoprotein morphology and structure were reviewed. MAJOR CONCLUSIONS: The comparisons show that an optimized NS (OpNS) protocol can eliminate the rouleau artifacts of lipoproteins, and that the lipoproteins are similar in size and shape as statistically measured from two EM methods, OpNS and cryo-electron microscopy (cryo-EM). OpNS is a high-throughput, high-contrast and high-resolution (near 1nm, but rarely better than 1nm) method which has been used to discover the mechanics of a small protein, 53kDa cholesterol ester transfer protein (CETP), and the structure of an individual particle of a single protein by individual-particle electron tomography (IPET), i.e. a 14Å-resolution IgG antibody three-dimensional map. GENERAL SIGNIFICANCE: It is suggested that OpNS can be used as a general protocol to study the structure of proteins, especially highly dynamic proteins with equilibrium-fluctuating structures.

Determining Macromolecular Structures Using Cryo-Electron Microscopy.

Structural insights into macromolecular and protein complexes provide key clues about the molecular basis of the function. Cryogenic electron microscopy (cryo-EM) has emerged as a powerful structural biology method for studying protein and macromolecular structures at high resolution in both native and near-native states. Despite the ability to get detailed structural insights into the processes underlying protein function using cryo-EM, there has been hesitancy amongst plant biologists to apply the method for biomolecular interaction studies. This is largely evident from the relatively fewer structural depositions of proteins and protein complexes from plant origin in electron microscopy databank. Even though the progress has been slow, cryo-EM has significantly contributed to our understanding of the molecular biology processes underlying photosynthesis, energy transfer in plants, besides viruses infecting plants. This chapter introduces sample preparation for both negative-staining electron microscopy (NSEM) and cryo-EM for plant proteins and macromolecular complexes and data analysis using single particle analysis for beginners.

Negative staining of lipopolysaccharides on polyacrylamide gels by using eosin B.

A sensitive and simple technique for the negative detection of lipopolysaccharides (LPSs) following polyacrylamide gel electrophoresis (PAGE) using eosin B (EB) was developed. After electrophoresis, gels were fixed, stained, and developed within 30 min to achieve transparent and colorless LPS bands under opaque gel matrix background. As low as 20 to 40 ng of total LPSs could be detected, which is 4-fold more sensitive than those of the widely used silver stain developed by Fomsgaard and coworkers and imidazole-zinc (IZ) negative stain. For its sensitivity and brevity, this stain may be a practical method for LPS determination in the routine laboratory.

Preyssler-type phosphotungstate is a new family of negative-staining reagents for the TEM observation of viruses.

Transmission electron microscopy (TEM) is an essential method in virology because it allows for direct visualization of virus morphology at a nanometer scale. Negative staining to coat virions with heavy metal ions must be performed before TEM observations to achieve sufficient contrast. Herein, we report that potassium salts of Preyssler-type phosphotungstates (K(15-n)[P5W30O110Mn+], M = Na+, Ca2+, Ce3+, Eu3+, Bi3+, or Y3+) are high-performance negative staining reagents. Additionally, we compare the staining abilities of these salts to those of uranyl acetate and Keggin-type phosphotungstate. The potassium salt of Preyssler-type phosphotungstates has the advantage of not requiring prior neutralization because it is a neutral compound. Moreover, the potassium counter-cation can be protonated by a reaction with H+-resin, allowing easy exchange of protons with other cations by acid-base reaction. Therefore, the counter-cations can be changed. Encapsulated cations can also be exchanged, and clear TEM images were obtained using Preyssler-type compounds with different encapsulated cations. Preyssler-type phosphotungstates may be superior negative staining reagents for observing virus. Polyoxotungstates (tungsten-oxide molecules with diverse molecular structures and properties) are thus promising tools to develop negative staining reagents for TEM observations.

Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples.

In recent years, electron cryo-microscopy (CryoEM) has become a powerful method for the high-resolution studies of biological macromolecules. While CryoEM experiments can begin without additional microscopy steps, negative-stain EM can tremendously minimize CryoEM screening. Negative-stain is a quick method that can be used to screen for robust biochemical conditions, the integrity, binding, and composition of samples and to get an estimation of sample grid concentration. For some applications, the map resolutions potentially afforded by stain may be as biologically informative as in CryoEM. Here, I describe the benefits and pitfalls of negative-stain EM, with particular emphasis on Uranyl stains with the main goal of screening in advance of CryoEM. In addition, I provide a materials list, detailed protocol and possible adjustments for the use of stains for biological samples requiring imaging and/or diffraction-based methods of EM.

Purification of Human Complement Component C4 and Sample Preparation for Structural Biology Applications.

Activated complement component C4 (C4b) is the nonenzymatic component of the classical pathway (CP) convertases of the complement system. Preparation of C4 and C4b samples suitable for structural biology studies is challenging due to low yields and complexity of recombinant C4 production protocols reported so far and heterogeneity of C4 in native sources. Here we present a purification protocol for human C4 and describe sample preparation methods for structural investigation of C4 and its complexes by crystallography, small angle X-ray scattering, and electron microscopy.

An optimized negative-staining protocol of electron microscopy for apoE4 POPC lipoprotein.

Apolipoprotein E (apoE), one of the major protein components of lipoproteins in the peripheral and central nervous systems, regulates cholesterol metabolism through its interaction with members of the low density lipoprotein receptor family. One key to understanding apoE function is determining the structure of lipid-bound forms of apoE. Negative-staining (NS) electron microscopy (EM) is an easy and rapid approach for studying the structure and morphology of lipid-bound forms of apoE. However, an artifact of using the conventional NS protocol is that the apoE phospholipid particles form rouleaux. In this study, we used cryo-electron microscopy (cryo-EM) to examine apoE4 palmitoyl-oleoylphosphatidylcholine (POPC) particles in a frozen-hydrated native state. By comparing the particle sizes and shapes produced by different NS protocols to those produced by cryo-EM, we propose an optimized protocol to examine apoE4 POPC particles. Statistical analysis demonstrated that the particle sizes differ by less than 5% between the optimized protocol and the cryo-EM method, with similar shapes. The high contrast and fine detail of particle images produced using this optimized protocol lend themselves to the structural study of lipid-bound forms of apoE.

Promoted negative staining of proteins in SDS-PAGE using Eosin B compounded with magnesium chloride.

In this study, we describe an effective visualizing technique for proteins in SDS-PAGE based on the organic dye, Eosin B, the sensitivity of which can be further strengthened by the addition of magnesium to the staining solution after electrophoresis. The newly developed protocol is low in cost and easily performed compared with the common methods for protein analysis in 1-D and 2-D gels. It provides a much better sensitivity (0.2 ng of single protein band) than that of imidazole-zinc negative stain for fixing and staining within 1 h, and an excellent performance in terms of compatibility with MALDI-TOF MS. The results show that similar identification scores and numbers of matched peptides were obtained by both methods. Furthermore, the effects of different metal salts on the quality of protein visualization by Eosin B were also investigated. Because of its sensitivity, stability, and safety, this stain may be a more practical method for protein determination in the routine laboratory.

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y.

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.

Zinc-imidazole negative staining of chromosomal-sized DNA molecules in agarose minigels.

We standardized the zinc-imidazole negative staining method for detecting chromosomal-sized DNA molecules separated by pulsed field minigel electrophoresis. The best experimental conditions were as follows: separating large DNA molecules in minigels of 0.4 cm thickness, further incubating them with 40 mM ZnSO4 solution, and finally incubating them with 0.1 and 2 M imidazole solutions successively. The lowest yeast cells/miniplug useful in DNA band detection was 3 x 10(7) cells, as occurred with ethidium bromide-stained minigel. Electrophoresis patterns were visualized as colorless bands contrasting against a white background after illuminating the minigel with white light. This negative staining method is nontoxic and preserves the chemical integrity of the DNA molecules.

LoTToR: An Algorithm for Missing-Wedge Correction of the Low-Tilt Tomographic 3D Reconstruction of a Single-Molecule Structure.

A single-molecule three-dimensional (3D) structure is essential for understanding the thermal vibrations and dynamics as well as the conformational changes during the chemical reaction of macromolecules. Individual-particle electron tomography (IPET) is an approach for obtaining a snap-shot 3D structure of an individual macromolecule particle by aligning the tilt series of electron tomographic (ET) images of a targeted particle through a focused iterative 3D reconstruction method. The method can reduce the influence on the 3D reconstruction from large-scale image distortion and deformation. Due to the mechanical tilt limitation, 3D reconstruction often contains missing-wedge artifacts, presented as elongation and an anisotropic resolution. Here, we report a post-processing method to correct the missing-wedge artifact. This low-tilt tomographic reconstruction (LoTToR) method contains a model-free iteration process under a set of constraints in real and reciprocal spaces. A proof of concept is conducted by using the LoTToR on a phantom, i.e., a simulated 3D reconstruction from a low-tilt series of images, including that within a tilt range of ±15°. The method is validated by using both negative-staining (NS) and cryo-electron tomography (cryo-ET) experimental data. A significantly reduced missing-wedge artifact verifies the capability of LoTToR, suggesting a new tool to support the future study of macromolecular dynamics, fluctuation and chemical activity from the viewpoint of single-molecule 3D structure determination.

Fast Small-Scale Membrane Protein Purification and Grid Preparation for Single-Particle Electron Microscopy.

The ongoing development of single-particle cryo-electron microscopy (cryo-EM) is leading to fast data acquisition, data processing, and protein structure elucidation. Quick and reliable methods to go from protein purification and optimization to grid preparation will significantly improve the reach and power of cryo-EM. Such methods would particularly constitute a tremendous advantage in structural biology of membrane proteins, whose published structures stay still far behind the number of soluble protein structures. Here we describe a fast, low-cost, and user-friendly method for the purification and cryo-EM analysis of a recombinant membrane protein. This method minimizes the amount of starting material and manipulation steps needed to go from purification to grid preparation, and could potentially be expanded to other membrane protein purification systems for its direct application in structure determination by single-particle cryo-EM.

Membrane Protein Cryo-EM: Cryo-Grid Optimization and Data Collection with Protein in Detergent.

Cryo-electron microscopy (cryo-EM) is a powerful tool for investigating the structure of macromolecules under near-native conditions. Especially in the context of membrane proteins, this technique has allowed researchers to obtain structural information at a previously unattainable level of detail. Specimen preparation remains the bottleneck of most cryo-EM research projects, with membrane proteins representing particularly challenging targets of investigation due to their universal requirement for detergents or other solubilizing agents. Here we describe preparation of negative staining and cryo-EM grids and downstream data collection of membrane proteins in detergent, by far the most common solubilization agent. This protocol outlines a quick and straightforward procedure for screening and determining the structure of a membrane protein of interest under biologically relevant conditions.

Viral Haufen are urinary biomarkers of polyomavirus nephropathy: New diagnostic strategies utilizing negative staining electron microscopy.

Haufen, i.e. discrete three-dimensional cast-like polyomavirus aggregates in the urine, mark polyoma BK-virus nephropathy (BKN) with positive and negative predictive values of greater than 95%. They are novel diagnostic biomarkers of BKN, an important infectious complication post kidney transplantation. Here, we describe technical details of Haufen detection by negative staining electron microscopy. We studied more than 400 urine samples from over 180 patients and developed easy-to-follow protocols for optimal specimen preservation and preparation, including sample clarification and concentration. We detail diagnostic clues to detect Haufen and illustrate pitfalls, including "Haufen-look-alikes," which can hamper the interpretation. Urinary Haufen detection by negative staining electron microscopy is a new application of an old, well-established diagnostic technique. The protocols described here are useful for pathologists and electron microscopists to search for Haufen in voided urine samples, to predict BKN noninvasively, and to help clinicians managing renal allograft recipients.

Optimized Negative-Staining Protocol for Lipid-Protein Interactions Investigated by Electron Microscopy.

A large number of proteins are capable of inserting themselves into lipids, and interacting with membranes, such as transmembrane proteins and apolipoproteins. Insights into the lipid-protein interactions are important in understanding biological processes, and the structure of proteins at the lipid binding stage can help identify their roles and critical functions. Previously, such structural determination was challenging to obtain because the traditional methods, such as X-ray crystallography, are unable to capture the conformational and compositional heterogeneity of protein-lipid complexes. Electron microscopy (EM) is an alternative approach to determining protein structures and visualizing lipid-protein interactions directly, and negative-staining (OpNS), a subset of EM techniques, is a rapid, frequently used qualitative approach. The concern, however, is that current NS protocols often generate artifacts with lipid-related proteins, such as rouleaux formation from lipoproteins. To overcome this artifact formation, Ren and his colleagues have refined early NS protocols, and developed an optimized NS protocol that validated by comparing images of lipoproteins from cryo-electron microscopy (cryo-EM). This optimized NS protocol produces "near native-state" particle images and high contrast images of the protein in its native lipid-binding state, which can be used to create higher-quality three-dimensional (3D) reconstruction by single-particle analysis and electron tomography (e.g. IPET). This optimized protocol is thus a promising hands-on approach for examining the structure of proteins at their lipid-binding status.

Recombinant Expression, Purification, and Assembly of p62 Filaments.

This chapter describes the recombinant overexpression of the canonical selective autophagy receptor p62/SQSTM1 in E. coli and affinity purification. Also described is the method to induce p62 filament assembly and their visualization by negative stain electron microscopy (EM). In cells, p62 forms large structures termed p62 bodies and has been shown to be aggregation prone. This tendency to aggregate poses problems for expression and purification in vitro, which is a prerequisite for structural analysis. Here, we describe the method to express and purify soluble p62, using the solubility tag, MBP, in conjunction with autoinduction. Furthermore, we describe the protocol to assemble p62 into filaments by controlling the ionic strength of its buffer, as well as the preparation of negative stain EM grids to visualize the filaments. In vitro formed p62 filaments can be used to study receptor cargo interactions in minimal reconstituted autophagy model systems.

High-resolution single-particle 3D analysis on GroEL prepared by cryo-negative staining.

Cryo-negative staining was developed as a complementary technique to conventional cryo-electron microscopy on supramolecular complexes. It allows imaging biological samples in a comparable state of structural preservation to conventional cryo-EM but the staining produces better contrast in accessible areas and allows data recording at lower defocus values. Cryo-negative staining vitrifies biological particles in the presence of a concentrated ammonium molybdate solution at neutral pH. It was successfully used to study the structure and dynamics of several macromolecules, such as human transcription factors and RNA polymerases. Imaging macromolecular complexes with cryo-negative staining has been established previously to better than 2 nm detail. However, it has not been verified so far whether cryo-negative staining also visualizes secondary structure elements. Using the well known E. coli GroEL chaperonin, we could show that the structure is well preserved to approximately 10 A resolution. Secondary structure details are at least partially resolved in the electron density map.

Negative staining across holes: application to fibril and tubular structures.

The negative staining technique, when used with holey carbon support films, presents superior imaging conditions than is the case when samples are adsorbed to continuous carbon films. A demonstration of this negative staining approach is presented, using ammonium molybdate in combination with trehalose, applied to several fibrillar and tubular samples. Fibrils formed from the amyloid-beta peptide and the protease inhibitor pepstain A spread very well unsupported across holes and the different polymorphic fibril forms can be readily assessed. However, tubular forms of amyloid-beta have a tendency to be flattened, due to surface tension forces prior to and during specimen drying. Sub-fibril assembly forms and D-banded rat tail type 1 collagen fibres are presented. The air-dried collagen images produced are shown to contain almost as much detail as those obtainable by cryo-negative staining. Fragile DNA and DNA-protein nanotubes are also shown to yield superior quality images to those produced on continuous carbon films. The iron-storage protein, frataxin, creates elongated oligomeric assemblies, containing bound ferrihydrite microcrystals. The iron particles within these flexuous oligomers can be defined in the presence of ammonium molybdate, but they are more readily demonstrated if the frataxin is spread across holes in the presence of trehalose alone. The samples used here serve to show the likely benefit obtainable from negative staining across holes for a range of other fibrillar and tubular samples in biology, medicine and nanobiotechnology.