Signal amplification in immunohistochemistry at the light microscopic level using biotinylated tyramide and nanogold-silver staining.

Signal amplification techniques greatly enhance the sensitivity of immunohistochemical (IHC) and in situ hybridization (ISH) methods. In particular, catalyzed signal amplification (CSA) using labeled tyramide or Nanogold-silver staining is an important signal amplification tool. We have applied a combination of both techniques, as has been introduced for ISH, for a further increase in sensitivity of an IHC method to detect cathepsin B. This lysosomal proteinase can also be expressed extracellularly, particularly in relation to cancer metastasis. Higher sensitivity of the IHC method was needed because existing methods failed to demonstrate cathepsin B protein where cathepsin B activity was found with a fluorescence enzyme histochemical method. Combined CSA and Nanogold-silver staining provided the sensitivity that was required. Moreover, this signal amplification method enabled the use of a 10-fold lower concentration of primary antibody (1 microg/ml). Nonspecific background staining was low provided that endogenous biotin, avidin, and peroxidase were completely blocked. The method was reproducible when all steps, and particularly the silver enhancement step, were rigidly controlled. The method resulted in localization patterns of cathepsin B protein that were in agreement with those of cathepsin B activity in serial sections of rat liver containing colon cancer metastases. We concluded that combined application of CSA and Nanogold-silver staining provides high sensitivity for immunohistochemical methods and that activity localization by an enzyme histochemical method is a very attractive alternative to IHC localization of an enzyme because it is at least as sensitive, it is rapid and simple, and it provides direct information on the function of an enzyme.     

An alkaline-phosphatase staining method in avidin-biotin immunohistochemistry

An avidin-biotin alkaline-phosphatase (ABAP) staining method has been developed for the labeling of tissue sections and cell smears. The introduction of alkaline phosphatase as a marker enzyme through an avidin bridge results in excellent immunocytochemical labeling of different antigens using poly- and monoclonal antibodies. This technique avoids problems with endogenous peroxidase activity that sometimes occur using peroxidase staining procedures. The introduction of a preformed avidin-biotin alkaline-phosphatase complex (ABAPC) makes the presented technique as simple to handle as the widely used avidin biotin-peroxidase complex method (ABC). The ABAPC technique could be combined with other enzymatic labelings for double immunoenzymatic staining.     

An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells

Intracellular staining is a widely used flow cytometry (FCM)-based technique to detect the expression of cytoslio nucleic antigens. However, intracellular staining of cells expressing cytosolic fluorescent protein (FP) markers was proven to be problematic as significant loss of the FP-signal was routinely observed. Using splenocytes harvested from mice constitutively expressing the enhanced yellow fluorescent proteins (YFP) as a model, we modified the widely used intracellular staining protocol and successfully achieved simultaneous detection of both the nuclear proteins and YFP in T-regulatory cells. The improved protocol can be used to perform antibody-based intracellular characterization of FP-labeled target cells, while maintaining their fluorescent reporter signals for easy tracing and identification.     

An improved polychrome staining method for thick epoxy sections.

Improved polychrome staining of 1-1.5 mum epoxy sections is achieved with sequential applications of a single basic fuchsin-methylene blue mixture at two different pH values. The dye solution is applied for 2-3 min at 50-52 C first at pH 7.9, then at pH 6.7. In sections of mouse mammary tissue, epithelial cells are stained deep blue, connective tissue pink, and fat cells bright olive-green. This simple technique consistently yields uniform, vivid, contrasting colors that sharply delineate the elements of the complex glandular architecture of the mammary gland. Similar polychromatic effects are obtained in applications to other tissues, such as stomach, adrenal gland, mammary tumor and artery.     

优化的细菌鞭毛染色技术

优化了实验教材上传统的银染液鞭毛染色方法,用单宁酸和FeCl3做媒染剂,增大单宁酸和FeCl3的质量浓度(并将其配制的溶液分别保存),然后用碱性染料沙黄水溶液[1]、齐氏石炭酸碱性复红染液[1]和稀释10倍吕氏碱性美蓝染液[1],分别对培养好的枯草芽胞杆菌进行染色,得到较粗、清晰的染色结果。     

An improved micropropagation protocol for teak

An improved micropropagation protocol has been developed for teak (Tectona grandis). Nodal explants placed on MS medium supplemented with 22.2 M benzylaminopurine and then serially transferred to fresh medium after 12, 24, 48 and 72 h gave maximum culture establishment (76.8%). Establishment was reduced when explants were retained in the initial culture medium longer than 12 h. Explants collected in May showed maximum (76.8%) response. Placement of the explants on MS medium supplemented with 22.2 M benzylaminopurine and 0.57 M indole-3-acetic acid resulted in the maximum average number of shoots. In vitro raised micro shoots were rooted ex vitro by dipping in indole-3-butyric acid (9.8 mM) for 2 min followed by planting in polyethylene pots containing a soil:vermiculite (1:1 v/v) mixture. This treatment resulted in 77.9% survival of the plantlets. They were weaned in a glasshouse and finally moved to an agro-net shade house.     

An improved immunofluorescence staining method for chloroplast proteins

Key message

An improved immunofluorescence staining method significantly facilitates the visualization of the subcellular localization of interested proteins in chloroplasts.

Abstract

As an important technical approach, immunofluorescence staining is widely used in the subcellular localization study of interested proteins. During the study of the functions of chloroplast division proteins, immunofluorescence staining was frequently adopted. Previously, a method has been developed to study the localization of a chloroplast division protein, FtsZ. However, it is laborious and time-consuming. In this study, we report a modified immunofluorescence staining method, in which protoplasts were isolated from leaf tissues, and then fixed for immunofluorescence staining. The time of the experiment was significantly reduced to several hours. Furthermore, we used correction pen in the fixation procedure and a new way to coat the slide, which greatly saved the cost of the experiment. With the chloroplast division protein ARC6 as an example, we can get a good fluorescence signal. Moreover, the localization of ARC6 in two chloroplast division mutants, arc3 and arc5, and three other plant species, such as cabbage, radish and pea, was also successfully analyzed with our new method. Overall, the immunofluorescence staining method we reported here is very practical, and it significantly facilitates the visualization of the subcellular localization of interested proteins in plant cells.

     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

An improved procedure for background correction in autoradiography

Summary In the event of weak autoradiographic labelling, the proportion of truly labelled cells or structures can be calculated from the frequency distributions of grains per area or cell structure fori=0, 1,...,n grains using the results obtained for an experimental group after the application of a radioactively labelled substance and those obtained for a control group without radioactivity. The principle of this computer-aided method is also applicable when the grain counts are related to varying areas in histological sections.Dedicated to Professor Dr. T.H.Schiebler on the occasion of his 65th birthday     

An improved procedure for background correction in autoradiography

In the event of weak autoradiographic labelling, the proportion of truly labelled cells or structures can be calculated from the frequency distributions of grains per area or cell structure for i = 0, 1,..., n grains using the results obtained for an experimental group after the application of a radioactively labelled substance and those obtained for a control group without radioactivity. The principle of this computer-aided method is also applicable when the grain counts are related to varying areas in histological sections.     

An improved P2P lookup protocol model

With the development of the peer-to-peer (P2P) technology, a lot of lookup protocols have come out. Although we can benefit from different protocols separately, our research shows that if there exists a proper model, most of the seemingly different protocols can be classified to a same framework. Since technology is a kind of human behavior, we can set up the P2P lookup general model referring to some outcomes of cognitive psychology. In this paper, we firstly study the lookup model in cognitive psychology, then introduce the model into P2P search strategy, at last we give an improved Chord arithmetic based on the binary tree.     

An improved silver staining procedure for schizodeme analysis in polyacrylamide gradient gels.

A simple protocol is described for the silver staining of polyacrylamide gradient gels used for the separation of restriction fragments of kinetoplast DNA [schizodeme analysis of trypanosomatids (Morel et al., 1980)]. The method overcomes the problems of non-uniform staining and strong background color which are frequently encountered when conventional protocols for silver staining of linear gels are applied to gradient gels. The method described has proven to be of general applicability for DNA, RNA and protein separations in gradient gels.