Loss of Silver Grains from Radioautographs Stained by Gallocyanin-Chrome Alum

Radioactive tissue sections covered with the film from Kodak Fine-Grain Autoradiographic Stripping Plate AR. 10 were stained with Ehrlich's hematoxylin or gallocyanin-chrome alum after exposure and photographic processing. Staining with gallocyanin-chrome alum at pH 1.7 and 2.4 dissolved the silver grains completely or almost completely in 1 to. Grains were quite visible after a 3 hr staining at pH 3.4, but a statistical analysis revealed a loss of grains, compared with unstained controls. Grains were also lost in slides immersed in solutions of gallocyanin alone at pH 2.5 for 24 hr but not in solutions of chrome alum alone, nor in some other alums. In sections stained 1 hr with Ehrlich's hematoxylin, the grains were not dissolved.     

A One-Step Immunostaining Method to Visualize Rodent Muscle Fiber Type within a Single Specimen

In this study, we present a quadruple immunostaining method for rapid muscle fiber typing of mice and rats using antibodies specific to the adult myosin heavy chain (MyHC) isoforms MyHC1, 2A, 2X, and 2B, which are common marker proteins of distinct muscle fiber types. We developed rat monoclonal antibodies specific to each MyHC isoform and conjugated these four antibodies to fluorophores with distinct excitation and emission wavelengths. By mixing the four types of conjugated antibodies, MyHC1, 2A, 2X, and 2B could be distinguished within a single specimen allowing for facile delineation of skeletal muscle fiber types. Furthermore, we could observe hybrid fibers expressing MyHC2X and MyHC2B together in single longitudinal muscle sections from mice and rats, that was not attained in previous techniques. This staining method is expected to be applied to study muscle fiber type transition in response to environmental factors, and to ultimately develop techniques to regulate animal muscle fiber types.     

一种制备鱼类染色体的新方法

自1966年Ojima建立鱼类染色体空气干燥法以来,鱼类染色体研究技术有了很大进展.目前有肾细胞培养法、上皮细胞培养法和外周血培养法等。细胞培养需要在有一定条件的实验室进行(如无菌操作),因而简便快速的制备鱼类染色体方法便不断产生,有PHA+秋水仙碱注射法、秋水仙碱体外注射法和CoCl+秋水仙碱体外注射等方法.本文介绍一种适合于在野外条件下进行的小型鱼类和鱼苗染色体制片方法。     

用家蝇取代果蝇制作唾腺多线染色体新技术

组建学生科技兴趣小组,对家蝇唾液腺染色体制片方法进行研究,开发了制片新技术,即加热处理家蝇三龄幼虫,然后用改进的缓冲液进行解离前预处理,再酸性解离、染色、观察、永久制片、显微照相.利用新方法,学生均能一次就获得细胞膜破裂、背景清晰、染色体分散良好、横纹清楚的制片.     

DNA from Maize with and without B Chromosomes: A Comparative Study

DNA preparations from 5B and 0B maize seedlings are indistinguishable in their buoyant density distribution in CsCl gradients. Their renaturation kinetics are identical at several stringency criteria. DNA competition studies fail to detect any component in 5B DNA redundant sequences which is lacking in 0B DNA. Homologous and heterologous duplexes formed between 5B and 0B DNA have virtually identical melting profiles. The DNA of B chromosomes is concluded to be very closely related to that of A chromosomes.     

A Rapid Method for Preparing High Quality Alizarin Stained Skeletons of Adult Mice

A simple three-day technique is described for preparing completely cleared and high quality alizarin stained total skeletons of adult mice. Unfixed specimens are partially macerated during staining. Older specimens are heated for 15 min in 1% KOH. A heated solution of benzyl and ethyl alcohol, glycerin, and water is used for final clearing and hardening. This procedure requires about 10 min work per specimen and greatly simplifies preparation of stained and cleared skeletons of adult mice. Another technique, giving slightly better preparations, but requiring 11-14 days, is also described.     

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called “CLARITY” was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.     

Detection of Plutonium Contamination in Humans by the Autoradiographic Method

The autoradiographic technique, using 5 μ paraffin sections in contact with 5 μ NTA emulsion for 10 hr. and similar sections of sputum for 3 days, was found to be more sensitive for detecting contamination with Pu²³⁹ than tests with a scintillation counter. Both human and pig skin were tested. The autoradiograms showed characteristic alpha tracks in the emulsion at sites of Pu deposition following its uptake either in solution or in particulate form. Autoradiography is recommended as a routine method to supplement information gained from chemical analyses and counting procedures.     

Handling Human Chromosomes by A Coumarin Technic

Small pieces of nonneoplastic and neoplastic human tissues, mainly from cervix uteri and soon after removal by biopsy, are transferred to a saturated aqueous solution of coumarin for 5-10 min, after which they are put into the regular aceto-carmine mixture for about 1 hr. A squash preparation is made with moderate pressure and the cover glass is sealed by a mixture made of equal volumes of Canada balsam and hard paraffin. These temporary preparations are suitable for the study of chromosomes for about 15 days, after which they can be made permanent by following one of the usual procedures that employ dehydration and clearing.     

一种检测人中期染色体原位切口移位的新方法

本研究首次详细描述了在BrdU替代4个细胞周期以上的中期染色体上进行原位染色体切口移位的方法。研究证明,切口移位效率达峰值的最适温度为15—20℃,最佳时间为10—15分钟,DNasc Ⅰ的最佳浓度为2ng/ml。用本方法进行的人外周血淋巴细胞染色体的原位切口移位带型表明,原位切口移位的染色体带型特征与已知的G带、R带有较明显的差异,是另一种新的带型。本方法较用’H-dTTP或Bio-dUTP作标记物进行染色体原位切口移位更简便、快速,不仅可用于活性基因在不同种类基因组内的分布研究,而且可能在细胞遗传学和分子生物学研究中具有更广泛的用途。     

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.     

Preclinical TSPO Ligand PET to Visualize Human Glioma Xenotransplants: A Preliminary Study

Current positron emission tomography (PET) imaging biomarkers for detection of infiltrating gliomas are limited. Translocator protein (TSPO) is a novel and promising biomarker for glioma PET imaging. To validate TSPO as a potential target for molecular imaging of glioma, TSPO expression was assayed in a tumor microarray containing 37 high-grade (III, IV) gliomas. TSPO staining was detected in all tumor specimens. Subsequently, PET imaging was performed with an aryloxyanilide-based TSPO ligand, [¹⁸F]PBR06, in primary orthotopic xenograft models of WHO grade III and IV gliomas. Selective uptake of [¹⁸F]PBR06 in engrafted tumor was measured. Furthermore, PET imaging with [¹⁸F]PBR06 demonstrated infiltrative glioma growth that was undetectable by traditional magnetic resonance imaging (MRI). Preliminary PET with [¹⁸F]PBR06 demonstrated a preferential tumor-to-normal background ratio in comparison to 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG). These results suggest that TSPO PET imaging with such high-affinity radiotracers may represent a novel strategy to characterize distinct molecular features of glioma growth, as well as better define the extent of glioma infiltration for therapeutic purposes.     

Shadow Casting of Stained Virus Bodies to Enhance Visibility by Light Microscopy

Materials used for study were viral smears or ultra-thin sections containing viral cell inclusions. They were stained with the Feulgen reaction and other cytochemical procedures. Stained preparations were dried and then shadow-cast with metallic chromium for 30 seconds in a bell jar with a vacuum of at least 0.1 µ (10^-4mm.) of mercury, and placed at a shadowing angle of 10-12°. Shadow-cast preparations were cleared with xylene and mounted in Canada balsam. Dried smears or deparaffinized sections without staining were suited to this method also. A virus which stained indistinctly with cytochemical procedures alone could be adapted to visible light microscopy by shadowing, and in addition, used for observations on its chemical composition.     

A Simple Drying Method for Field Emission Scanning Electron Microscopy for Chromosomes

Hexamethyldisilazane treatment and subsequent air drying of spread plant chromosomes is compared with critical point drying. The two procedures are equivalent for preparing chromosomes for examination by field emission scanning electron microscopy at low voltage.