Standardization in Biological Staining. The Influence of Dye Manufacturing

The purpose of biological staining is to obtain specimens of biological material that can be assessed in the microscope. These specimens are influenced by all processes from removal from the intact organism to mounting on the microscopic slide. To achieve comparable results with various techniques for biological staining, standardization of all procedures and reagents is mandatory. In this paper, I focus particularly on dyes and consider the possibilities for obtaining standardized dyes. In general practice, most biological staining takes place with available commercial dyes. These dyes may or may not have been subjected to quality assessment either internally by the producer or vendor or externally by independent investigators or organizations such as the Biological Stain Commission. Concerted attempts at standardization in Europe are discussed. The latest results of this work, the European standard EN 12376, is presented. This standard is concerned with information supplied by the manufacturer with in vitro diagnostic reagents for biological staining. The standard has been prepared by a Working Group on Staining in Biology under Technical Committee 140, In Vitro Medical Devices, of the European committee for standardization, CEN.     

Amyloid from a histochemical perspective. A review of the structure,properties and types of amyloid,and a proposed staining mechanism for Congo red staining

Amyloid is a diverse group of unrelated peptides or proteins that have positive functionality or are associated with various pathologies. Despite vast differences, all amyloids share several features that together uniquely define the group. 1) All amyloids possess a characteristic cross-ß pattern with X-ray diffraction typical of ß-sheet secondary protein structures. 2) All amyloids are birefringent and dichroic under polarizing microscopy after staining with Congo red, which indicates a crystalline-like (ordered) structure. 3) All amyloids cause a spectral shift in the peak wavelength of Congo red with conventional light microscopy due to perturbation of π electrons of the dye. 4) All amyloids show heightened intensity of fluorescence with Congo red, which suggests an unusual degree of packing of the dye onto the substrate. The ß portion of amyloid molecules, the only logical substrate for specific Congo red staining under histochemical conditions, consists of a stack of ß-sheets laminated by hydrophilic and hydrophobic interactions between adjacent pairs. Only the first and last ß-sheets are accessible to dyes. Each sheet is composed of numerous identical peptides running across the width of the sheet and arranged in parallel with side chains in register over the length of the fibril. Two sets of grooves are bordered by side chains. X grooves run perpendicular to the long axis of the fibril; these grooves are short (the width of the sheet) and number in the hundreds or thousands. Y grooves are parallel with the long axis. Each groove runs the entire length of the fibril, but there are very few of them. While Congo red is capable of ionic bonding with proteins via two sulfonic acid groups, physical constraints on the staining solution preclude ionic interactions. Hydrogen bonding between dye amine groups and peptide carbonyls is the most likely primary bonding mechanism, because all ß-sheets possess backbone carbonyls. Various amino acid residues may form secondary bonds to the dye via any of three van der Waals forces. It is possible that Congo red binds within the Y grooves, but that would not produce the characteristic staining features that are the diagnostic hallmarks of amyloid. Binding in the X grooves would produce a tightly packed series of dye molecules over the entire length of the fibril. This would account for the signature staining of amyloid by Congo red: dichroic birefringence, enhanced intensity of fluorescence and a shift in visible absorption wavelength.     

DNA staining in agarose and polyacrylamide gels by methyl green

ABSTRACT

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G? (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.     

Evolution of the silver and gold stains in neurohistology

Scientific investigations depend on the reliability of the observations that can be made. This reliability is determined in part by the understanding of the techniques and technology used to make the observations. The limitations and the strengths of the methodology and the equipment used must be evaluated thoroughly. The extent to which this is and has been the case for the use of the metal based stains in neuroscience is the subject of this paper. I evaluate the metallic stains used for neuroscience from several perspectives. I review briefly the state of neurohistology prior to its “golden years,” 1870–1910. Then I trace the development of the silver based stains used for neurohistology. I wanted to discuss the reasoning used by the originators of the silver based techniques in developing their specific procedures, but discovered that while procedures may be published, the methods and ideas used to arrive at the final procedures are not usually described in published work.     

Monte Carlo Study of the Buckingham Exponential-six Fluid

Abstract

In this paper we report the results of extensive Monte Carlo simulations of a pure fluid of Buckingham modified exponential-six molecules. Data are presented for the configurational energy and pressure covering a wide range of temperatures and densities. These data are interpreted using the generalized van der Waals partition function with a novel separation into free volume and mean potential terms. We find, surprisingly, that the Buckingham fluid is described by a simple van der Waals-like equation of state provided that the b parameter is temperature dependent and chosen in a theoretically correct manner.     

Three-Dimensional Structure of CAP-Gly Domain of Mammalian Dynactin Determined by Magic Angle Spinning NMR Spectroscopy: Conformational Plasticity and Interactions with End-Binding Protein EB1

Microtubules and their associated proteins play important roles in vesicle and organelle transport, cell motility and cell division. Perturbation of these processes by mutation typically gives rise to severe pathological conditions. In our efforts to obtain atomic information on microtubule-associated protein/microtubule interactions with the goal to understand mechanisms that might potentially assist in the development of treatments for these diseases, we have determined the three-dimensional structure of CAP-Gly (cytoskeleton-associated protein, glycine-rich) domain of mammalian dynactin by magic angle spinning NMR spectroscopy. We observe two conformations in the β2 strand encompassing residues T43-V44-A45, residues that are adjacent to the disease-associated mutation, G59S. Upon binding of CAP-Gly to microtubule plus-end tracking protein EB1, the CAP-Gly shifts to a single conformer. We find extensive chemical shift perturbations in several stretches of residues of CAP-Gly upon binding to EB1, from which we define accurately the CAP-Gly/EB1 binding interface. We also observe that the loop regions may exhibit unique flexibility, especially in the GKNDG motif, which participates in the microtubule binding. This study in conjunction with our previous reports suggests that conformational plasticity is an intrinsic property of CAP-Gly likely due to its unusually high loop content and may be required for its biological functions.     

Dynamic expression of miR-126 and its effects on proliferation and contraction of hepatic stellate cells

In our previous study, miR-126 was identified as one of the leading miRNAs that is downregulated during activation of hepatic stellate cells (HSCs). However, the roles and related mechanisms of miR-126 in HSCs are not understood. In this study, we compared expression of miR-126 during HSC activation both in vitro and in vivo. We also applied RNA interference to analyze the role and mechanism of miR-126^∗ in the activation of HSCs. Restoring HSCs with Lv-miR-126^∗ resulted in decreased proliferation, accumulation of extracellular matrix components, and cell contraction, while also negatively regulating the vascular endothelial growth factor (VEGF) signal transduction pathways by partially targeted VEGF-A. Thus, we postulate that miR-126 may be a biological marker for the activation of HSCs, and useful for reducing intrahepatic vascular resistance and improving the sinusoidal microcirculation in chronic liver diseases.     

白桦反义CCoAOMT基因调控木质素生物合成

白桦是我国北方重要的造林树种,但其中的高木质素含量严重制约了它作为造纸资源植物的开发利用。本文利用RACE技术获得了白桦咖啡酰辅酶A-3-O-甲基转移酶（CCoAOMT）基因全长ORF序列,并构建了白桦CCoAOMT基因的反义表达载体,通过农杆菌介导法将其导入到白桦中。PCR检测表明反义CCoAOMT基因已整合到白桦的基因组中。对转化植株的半定量PCR检测显示转基因株系的CCoAOMT基因表达量下降;Wiesner染色发现,与野生型相比,转基因植株木质素含量有所下降。对七年生的转基因白桦和野生型对照进行了化学成分分析,结果表明转基因白桦的苯醇抽提物和Klason木质素显著减少,聚戊糖含量升高。上述结果暗示BpCCoAOMT基因参与白桦木质素的合成,反义表达该基因后木质素含量减少,更易于去除。白桦CCoAOMT基因对木质素的合成起重要作用,这为培育低木质素含量的制浆新品种白桦奠定了基础。     

Synthesis and elaboration of N-methylpyrrolidone as an acetamide fragment substitute in bromodomain inhibition

N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.