Thin Histological Sections Prepared from Large Thick Sections: a New Technique

A method is described for obtaining thin (1 μm) sections for light microscopy from large area thick (100 μm) sections of low viscosity nitrocellulose embedded specimens of human spinal osteoligamentous material.     

Stained Ground Sections of Teeth and Bone

Formalin-fixed rat hemimandibles were ground to approximately 80μ with the aid of a diamond disc attached to a dental handpiece. A 1/10-hp motor attached to the handpiece by a pulley powered the disc to speeds up to 4000 rev/min. Contact between the hemimandible, which was placed on water-moistened cork, and the diamond disc was made possible through manipulation of the handpiece. The ground sections obtained by this procedure were stained with hematoxylin and eosin and periodic acid-Schiff for microscopic study. The fine structure of mineralized tissues (teeth and bones) as well as related soft structures, e.g., blood vessels, endosteum, pulp, etc., were demonstrated. The procedure did not require the embedding of the tissue nor were complicated grinding devices necessary.     

Effects of Sunlight on Stained Sections Mounted in Various Media

Sections stained with haematoxylin and eosin showed maximum optical density (OD) at 536 nm, with a second peak at 600 nm. Sections stained with only eosin showed a peak at 536 nm, whilst those stained with haetnatoxylin showed a peak at 600 nm. Reduction in OD at these wavelengths was used to estimate fading of the staining. Direct sunlight reduced the OD of sections mounted in 22 different mounting media by 14 to 64% at 536 nm and 12 to 51% at 600 nm.     

Giemsa Poststaining of Sections Stained for Acetyl-Cholinesterase Improves Sensitivity and Contrast

During investigations into the cholinergic innervation of blood vessels in skeletal muscle, it was found that poststaining of sections with Giemsa's stain (Gurr, R66) after incubation to reveal acetylcholinesterase activity as copper ferrocyanide (El-Badawi and Schenk 1967) not only produced nuclear and cytoplasmic counter-staining, but also resulted in intensification of the reaction, resulting in deep blue-black nerve endings (Fig. 1). Areas previously distinguishable only by phase contrast were easily recognizable after Giemsa staining. The method described was originally used on 10 μm cryostat sections, pre- or postfixed in formolcalcium and stained in the reaction mixture described by El-Badawi and Schenk (1967) for 30-60 minutes. After rinsing in distilled water (5 min), sections were stained in Giemsa's stain (3 min), washed well in distilled water, rapidly dehydrated, cleared and mounted.     

Prevention of Negative Chemography in Phenylenediamine Stained Autoradiographs of Plastic Semithin Sections

Negative chemography is the loss of latent image daring autoradiographic exposure, due to reactive groups in the specimen. Tissue fixed with ghuaraldehyde and osmium tetroxide, block stained with ft-phenylenediamme and embedded in Epou for light microscope sections causes intense negative cbemography when autoradiographed by dipping in Ilford K2 emulsion: this cannot be completely prevented by separating section from emulsion by means of a layer of evaporated carbon. Chemical treatment of the sections before autoradiography may reduce the cbemography. Treatment with 1 % hydrogen peroxide for 15 nun reduced it to such an extent that subsequent coating with 5 nm carbon abolished it. Material block stained in this way gave excellent contrast, both for light and electron microscopy.     

Paraffin Sections of Nervous Tissue Supra Vitally Stained with Methylene Blue: A New, Reliable and Simple Fixation Technique

TWO steps are recommended for fixation of central nervous tissue after supravital staining with methylene blue: application of ammonium heptamolybdate followed by a paraformaldehyde-glutaraldehyde mixture. The two fixatives complement each other so that both dye and tissue are optimally fixed. Molybdate renders the dye water and alcohol insoluble, the aldehyde mixture conditions the tissue for embedding in paraffin. This technique is a suitable alternative to conventional whole mount and frozen sectioning methods.     

Spectrally Resolved Morphometry of the Nucleus in Hepatocytes Stained by Four Histological Methods

A novel concept of spectrally resolved morphometry for histological specimens was developed using light microscopy combined with spectrally resolved imaging. The spectroscopic characteristics of rat hepatocytes stained by Haematoxylin and Eosin, Romanowsky–Giemsa, periodic acid–Schiff and Masson's trichrome were assessed. Light intensity in the range 450–850 nm was recorded from 10000 pixels of nuclear domains of each stained cell and represented as light transmittance spectra and optical density. In order to identify spectral shifts caused by stain– macromolecule interactions, we compared the spectra of individual stain components with those of DNA and bovine serum albumin. Chromatin and interchromatin areas were classified spectrally using a chosen spectral library followed by morphometric calculations of nuclear domains for each staining method. The spectral fingerprints of Masson's trichrome stain distinguished the nucleolus from the rest of the nuclear c hromatin, enabling the demarcation and calculation of the nucleolar area. Spectrally resolved imaging of human hepatocytes stained by Masson's trichrome stain revealed marked differences between the nucleolar area in normal human hepatocytes compared with hepatocellular carcinoma. Masson's trichrome stain also distinguished the nucleolar area in human breast carcinoma cells and keratinocytes.     

Application of the Papanicolaou Stain to Paraffin Sections

Routine paraffin sections from tissues fixed either in aqueous formalin, 80% alcohol (with or without 1% trichloracetic acid added), Carnoy's alcohol-chloroform-acetic (6:3:1) and Bouin's fixative were stained as follows: Harris' hematoxylin, 6 min; running water, 2-3 min; ascending grades of alcohol to 95%; orange G, 0.5% and phosphotungstic acid, 0.015% in 95% alcohol, 5 min; 95% alcohol, 2 changes; Papanicolaou's EA36, 2.5 min; dehydration, clearing, and covering in Permount. The results show morphology better than hematoxylin and eosin and the technic is recommended particularly for keratin, which always stains bright orange.     

Method for Histological Preparation of Bone Sections Containing Titanium Implants

A thin sectioning technique involving hand grinding has been developed to produce 20—40-μn-thick sections of bone-titanium implant sites. Components include: 1) surface staining of sections prior to mounting on slides so bone labels (oxytetracycline-HCI and 2,4-bis(N,N-dicarbometnyl) aminomethylfluorescein (DCAF)) can be seen in sections viewed with transmitted light, 2) a pneumatic sample press for bonding sections to slides with a thin, uniform glue line and without trapped air bubbles, and 3) bonding methyl methacrylate embedded sections to clear acrylic slides with methyl methacrylate monomer to provide enhanced bond strength and grinding properties compared to those obtainable with glass slides. Sample cracking and distortion is minimized and the tissue-implant interface can be kept intact The expense of start-up equipment for this technique is minimal.     

The Application of Miller's Elastic Stain to Glycol Methacrylate Tissue Sections

The application of Miller's dilute elastic stain followed sequentially by Gill's III hematoxylin and a fast green counterstain produced a reliable and consistent method for differentially staining elastic fibers, nuclei, muscle and collagen in glycol methacrylate tissue sections. Evaluation of different methods of fixation and conditions of staining on animal tissue sections showed that elastic fibers in both perfusion and immersion fixed tissues can be intensely stained. The stability of Miller's elastic stain offers the potential of a commercially available histological stain reagent for coarse and fine elastic fibers in glycol methacrylate tissue sections.     

Improved Techniques Using Giemsa Stained Glycol Methacrylate Tissue Sections to Quantitate Basophils and Other Leukocytes in Inflammatory Skin Lesions

Improved techniques were developed for processing inflammatory skin lesions in glycol methacrylate (JB-4, Polysciences, Inc.) and for quantitating their leukocyte infiltrates by light microscopy: (1) fixation of entire pelts from rabbits, guinea pigs, rats and mice bearing multiple lesions eliminated artifacts due to biopsy and produced uniformly oriented skin sections; (2) adding dimethylsulfoxide and hydrogen peroxide to the Karnovsky-type fixative increased the rate and effectiveness of fixation; (3) the presence of glycerol in the infiltrating methacrylate and the polymerized plastic block improved the sectionability of skin and other tissues; (4) coating slides with JB-4 Solution A prevented detachment of specimens; (5) Giemsa staining at a carefully selected pH provided optimal differentiation of leukocytes from the several species examined, including man. These techniques, which allowed an accurate histologic assessment of inflammatory skin lesions, were especially valuable for quantitating basophils.     

Translucent Tray Apparatus for Rapid Scanning of Serial Histological Sections in Volume

The simple apparatus described combines a binocular stereoscopic microscope and support stand, an adaptation of an x-ray viewing box, and a lucite tray modification of histological storage trays to permit rapid scanning of serial histological sections.     

The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

A Method for Histological Preparation of Undecalcified Bone Sections Containing Acrylic Bone Cement

An improved and time reducing method is presented for the histological evaluation of bone containing polymethylmethacrylate (PMMA) bone cement. The undecalcified bone was embedded in epoxy resin and sections of 50-100 μm thickness were produced using a commercially available cutting grinding system. The sections were stained with Stevenel's blue and van Gieson picrofuchsin or a modified hematoxylin-eosin. PMMA bone cement was not dissolved and remained enabling examination in situ of an intact cement bone interface and tissue reaction without decalcification.     

药物制剂技术在基因药物研制中的应用

基因药物是未来药物的发展方向,必须研究适宜的给药系统以促进基因药物的吸收和控制药效。基因药物的给药途径主要包括注射、口服、肺靶向、脑靶向和心血管基因转换等。所采用的剂型主要包括微球、脂质体、微乳等。本文就基因药物给药途径、所采用的主要剂型及各自所具备的优势作一综述。另外,简要介绍了基因药物给药系统的纳米技术。