Enrichment of Bruch's Membrane from Human Donor Eyes

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the developed world. The disease manifests itself by the destruction of the center of the retina, called the macula, resulting in the loss of central vision. Early AMD is characterised by the presence of small, yellowish lesions called soft drusen that can progress onto late AMD such as geographic atrophy (dry AMD) or neovascularisation (wet AMD). Although the clinical changes are well described, and the understanding of genetic influences on conferring AMD risk are getting ever more detailed, one area lacking major progress is an understanding of the biochemical consequences of genetic risk. This is partly due to difficulties in understanding the biochemistry of Bruch’s membrane, a very thin extracellular matrix that acts as a biological filter of material from the blood supply and a scaffold on which the retinal pigment epithelial (RPE) cell monolayer resides. Drusen form within Bruch’s membrane and their presence disrupts nutrient flow to the RPE cells. Only by investigating the protein composition of Bruch’s membrane, and indeed how other proteins interact with it, can researchers hope to unravel the biochemical mechanisms underpinning drusen formation, development of AMD and subsequent vision loss. This paper details methodologies for enriching either whole Bruch’s membrane, or just from the macula region, so that it can be used for downstream biochemical analysis, and provide examples of how this is already changing the understanding of Bruch’s membrane biochemistry.     

An Improved Method of Decalcification

Following several experimental investigations, an improved method of decalcification has been devised. The principle of this decalcification method is to obtain complete decalcification by a mixture of as high pH as possible without diminishing the stainability of the Nissl-granules (with Einarson's progressive staining method by means of gallocyanin). This is accomplished by the help of a buffer solution of equal parts of 8 N formic acid and 1 N sodium formate (pH 2.2). After-treatment consists only in rinsing in flowing water for 24 hours. Dehydration is in alcohol (70%, 96%, 100%); cedar oil; ligroin. Embedding in paraffin follows.     

An Improved Method of Decalcification

Following several experimental investigations, an improved method of decalcification has been devised. The principle of this decalcification method is to obtain complete decalcification by a mixture of as high pH as possible without diminishing the stainability of the Nissl-granules (with Einarson's progressive staining method by means of gallocyanin). This is accomplished by the help of a buffer solution of equal parts of 8 N formic acid and 1 N sodium formate (pH 2.2). After-treatment consists only in rinsing in flowing water for 24 hours. Dehydration is in alcohol (70%, 96%, 100%); cedar oil; ligroin. Embedding in paraffin follows.     

明胶酶谱实验方法的优化简

目的：优化明胶酶谱实验方法,并检测不同浓度凝血酶刺激人皮肤成纤维细胞分泌的明胶酶B（MMP-9）活性的变化。方法：用含明胶的聚丙烯酰胺凝胶电泳分开上清中各蛋白条带,经2．5％TritonX-100洗脱、明胶酶缓冲液孵育、考马斯亮蓝染色液染色及脱色液脱色后,经光密度扫描记录,应用ImageJ软件进行密度计量分析。结果：凝血酶刺激人皮肤成纤维细胞分泌MMP一9具有浓度依赖性。结论：利用优化的明胶酶谱实验方法可做出高质量的明胶酶谱图片,对检测明胶酶活性有重要意义。     

An Improved Arginine Histochemical Method

An improved arginine histochemical method is described. Development of the color at ice-bath temperature and other modifications produce more color. Uniformity of color development is obtained by transferring the slides quickly so that one solution does not drain from the slide before immersion in the next solution. Clearer sections are obtained by using a tetraethylammonium hydroxide solution to wash out the inorganic alkali, which is potassium hydroxide in the improved method. More stability of color is obtained by mixing tetraethylammonium hydroxide with the mounting medium.     

组氨酸生产中间控制方法的研究

研究了组氨酸与Pauly试剂显色反应的适宜条件 ,并加入钠盐和酪氨酸对标准曲线进行校正后 ,作为组氨酸定量测定的工作曲线。该方法简便 ,快速 ,准确度高 ,重现性好     

An Improved Method for Standardization of Microspectrofluorometers

The use of meshed cadmium-zinc sulfide fluorescent particles in combination with an electron microscope locator grid greatly facilitates the standardization of miam spectrofluorometers. The fluorescent particles emit maximally at 570 nm, and the intensity of fluorescence is directly proportional to cross-sectional area when epi-illumination is used Details concerning technique and fluorescent particle characteristics are reported.     

钙调素依赖性环核苷酸磷酸二酯酶的简易粗提法

<正> 钙调素依赖性环核苷酸磷酸二酯酶(camodulindependent cyclic nucleotide phosphodiesterase, CaM-PDE)是研究环核苷酸和钙调素酶法测定必备的工具。由于其稳定性较差,不能长期保存,需经常提取。传统提取方法采用冷室中线性离子梯度DEAE纤维素柱层析与其它层析并用。本法利用DE_(52)柱层析,阶段离子浓度洗脱。纯化CaM-PDE近20倍。在足够的钙调素(CAM)存在下可激活10倍。     

An Improved Method for Estimating Macromolecular Synthesis

Estimating rates of DNA, RNA, and protein synthesis has beengreatly facilitated by the use of radioactive precursors. Currentlyavailable methods to assess macromolecular synthesis in higherplants are time-consuming and imprecise. In the procedure outlinedhere, the conventional method is simplified by processing thetissue intact. This method allows a rapid analysis and considerablyreduces the inherent variability associated with the conventionalmethod.     

一种改进的蛋白质超薄凝胶电泳方法

介绍了一种将聚丙烯酰胺凝胶固定在电泳夹板上的蛋白质电泳方法.通过此方法蛋白质电泳可以在0.4 mm厚的聚丙烯酰胺凝胶上进行.实验证明,经此方法处理的玻板结合凝胶非常牢固,在电泳后的所有处理步骤中都不会发生凝胶脱落现象.     

An Improved Method for the Study of Chromosomes

It has been felt strongly that the current methods for the fixation of chromosomes are inadequate, inasmuch as the reagents are unduly long in reaching the important elements of the cells, particularly the nucleus. A realization of this situation has been at the bottom of the development of the so-called smear method. This method can, however, be advantageously employed only where loose cells are available, as in the reproductive elements of plants and animals. The present author has found that cutting up the material in very thin slices of almost microscopical tenuity, and passing them immediately into the best fixing solutions, namely those containing osmic acid, gives excellent results. The material is then assembled on cards in accordance with the present author's mass method (for which see the original article), and after embedding in nitrocellulose is sectioned 5μ or even thinner. Practically all methods of staining may be used, but the most satisfactory results have been achieved with Heidenhain's hematoxylin bleached almost to disappearance and followed by prolonged treatment with an aqueous safranin. This differentiates the chromosomal structures extremely well. It is clear by this method that chromosomes in all cases consist of a ground substance in which are situated two chromatids. In the somatic tissues these chromatids are apparently generally spiral and run in opposite directions. The crossing points of these spirals are responsible for the optical illusion which has been designated the gene.     

An Improved Method for the Synthesis of Flavanones

An isomerization of 2"-hydroxychalcones into the corresponding flavanones in ethanol in the presence of triethylamine is described.     

An Improved Method for Marine Cyanobacterial DNA Isolation

Summary The method of Bolch, Blackburn, Jones, Orr & Grewe [Phycologia, 36, 6 11, 1997] developed for isolation of DNA from freshwater cyanobacteria was suitably modified to yield a simple, efficient and reproducible protocol for the isolation of DNA from different morphological types of marine cyanobacteria. This method resulted in a high yield of quality DNA suitable for polymerase chain reaction (PCR) amplifications.     

An Improved Method for Studying Grass Leaf Epidermis

Leaf epidermis of grasses is elaborate and important in the systematica of the Poaceae at subfamily and genus level. Most available techniques used in preparing leaf epidermis for microscopic studies are time-consuming and often produce preparations inadequate for studying histological detail. A combination of the hand scraping and maceration methods with modifications is proposed in this paper to prepare epidermal peels comparatively rapidly. One epidermal layer was scraped off and the mesophyll tissue removed from the epidermis to be studied by maceration in HNO, The recovered epidermal peel was neutralized in NaOH and stained with malachite green or safranin O. Preparations made by this technique are suitable for studies of epidermal features, measurements of special structures and determinations of trichome indices. This method has been used in a study investigating intraspecific variation in southern African pasture grasses.     

一种改进的cDNA文库固相构建法

本文介绍一种新的cDNA文库固相构建法。文库构建过程中,cDNA的合成和修饰全部在固相介质――磁珠上完成,简便、迅速、文库质量高,能满足不同目的的cDNA文库构建,是一种值得借鉴和应用的好方法。 Abstract：A method for cDNA library construction was introduced.All enzymatic steps of library synthesis were performed on a solid support-magnetic beads.Therefore it combines fast speed and convenient with high quality library,making it ideally suited for most purposes.It is a good method and worth learning and utilization.     

An Improved Method for Preparing Anti-B Lymphocyte Serum

THE preparation of a heterologous antiserum specific to the bursa equivalent (B) lymphoid cells of the mouse requires extensive absorption with mouse tissues, especially thymocytes, to obtain specificity. The cell surface antigen(s) against which the serum reacts has been termed mouse specific bone marrow derived lymphocyte antigen(s) (MBLA)¹. The specificity of anti-MBLA for the B lymphoid population has been demonstrated by cytotoxicity tests, by its ability to inhibit a portion of antigen binding cells and by adoptive immune responses of antiserum purified lymphoid cells (refs. 1 and 2 and unpublished results of E. Möller and myself).