A Membrane Filter-Giemsa Procedure for Yeast Microcolonies

Yeast mlcrocolonies grown on 5 × 10 nun rectangles of membrane (Millipore) filter are treated through the membrane pores with 0.5% sodium azide for 2-3 hr, 10% perchloric acid for 4 days, and Giemsa stain for 1 day. After dehydration in air, they are permanently mounted. Similar preparations can be made with cells from sporularion cultures.     

Double Embedding Broad Thin Leaves in Paraffin

Paraffin pellets were melted in 24 × 24 × 5 mm stainless steel base molds. Specimens of leaves, 18 × 18 mm, were fixed, dehydrated and infiltrated with paraffin. Two specimens were transferred into molten paraffin on their laminar surfaces in a base mold and moved quickly onto a cold surface to cast them in a shallow block of paraffin. Each block was then scored with a razor blade, broken into two primary blocks, and trimmed to 20 × 9 mm with 5 mm flat edges. Each primary block was immersed upright on its long edge in a 22 × 22 × 20 mm Peel-A-Way® embedding mold containing molten paraffin. The leaf edge was held centrally in the mold while moving the double embedment onto a cold surface. In this secondary block, the leaf specimen stood perpendicular to the sectioning surface in perfect orientation for transverse ribbon sectioning. The two phases of paraffin bonded well.     

Chemical Dehydration of Specimens with 2,2-Dimethoxypropane (DMP) for Paraffin Processing of Animal Tissues: Practical and Economic Advantages over Dehydration in Ethanol

Chemical dehydration can be accomplished using 2,2-dimethoxy-propane (DMP). In the presence of an acid catalyst, this liquid reacts with water generating methanol and acetone as products. Although DMP is more expensive per milliliter than ethanol and other solvents used for dehydration, it is an economical alternative because a much smaller volume is needed. Slow penetration of DMP was previously thought to restrict its use to tiny specimens, but we now show that pieces of tissue as thick as 2 cm are dehydrated by overnight immersion in acidified DMP. We also show that dehydration in acidified DMP does not impair the staining of UNA or other basophilic components of animal tissues. The temperature and concentrations of methanol and H⁺ in the chemical dehydrating agent are too low to produce histochemicaUy detectable methylation or nucleic acid extraction.     

Acridine Orange Fluorescence in Paraffin Sections

The technique of staining with acridine orange for fluorescence microscopy of fresh animal and plant cells, chiefly for the detection of ribonucleic acid in the cytoplasm, was brought to a high degree of perfection by Schümmelfeder (1950) and has been developed further by Bertalanffy and Bickis (1956). Its employment for cancer detection in smears was reviewed by Bertalanffy, Masin and Masin in 1956.     

Two Methods for Flat Embedding Sections in LR White Cut from Paraffin Blocks

Two simple methods are described for flat embedding of sections cut from paraffin blocks of brain tissue in the hydrophilic resin LR White. The ability of fresh resin to polymerize when added to already cured resin is exploited. In the first method, a tissue section with a drop of LR White is pressed to the bottom of a gelatin capsule using a blank block. In the second method, the section in a drop of resin is sandwiched between the sawed halves of a blank block. After curing, thin sections containing large areas of tissue can be collected and processed for immunocytochemistr y.     

Rapid Dehydration for Celloidin Embedding by Means of Azeotropic Distiliation

By including plant material in a fractional distillation apparatus containing 95% alcohol and benzene it is possible to eliminate water completely in a constant boiling mixture. The dehydrated material can then be embedded in celloidin by orthodox techniques. In spite of the rapidity of the dehydration quite soft tissue as well as woody material can be processed. The method is cheap because it does not require absolute alcohol and is also suited to the humid climate in which it was developed.     

Repetitive Somatic Embryogenesis of Ocotea catharinensis Mez. (Lauraceae): Effect of Somatic Embryo Developmental Stage and Dehydration

Repetitive embryogenesis of Ocotea catharinensis from globular/early cotyledonary somatic embryos was successfully supported by WPM supplemented with 22.7 g l⁻¹ sorbitol, 20 g l⁻¹ sucrose, 400 mg l⁻¹ glutamine and 2 g l⁻¹ Phytagel. The best medium to induce repetitive embryogenesis in cotyledonary somatic embryos was half strength WPM supplemented with 20 g l⁻¹ sucrose, 400 mg l⁻¹ glutamine, 1.5 g l⁻¹ activated charcoal and 2 g l⁻¹ Phytagel. The mature somatic embryos gradually air dehydrated showed repetitive embryogenesis after subculture on half strength B5 medium supplemented with 20 g l⁻ sucrose, 20 g l⁻¹ Phytagel, 1.5 g l⁻¹ activated charcoal, 115.6 μM gibberellic acid and 214.8 μM naphthaleneacetic acid. The early cotyledonary, cotyledonary and mature somatic embryos tolerated respectively 95, 86 and 54% fresh weight losses without losing their repetitive embryogenesis potential. Cotyledonary and mature somatic embryos gradually air dehydrated in sealed Petri dishes showed 40–41% repetitive embryogenesis respectively after 20 days and 12 weeks desiccation storage. Repetitive embryogenesis in cotyledonary somatic embryos was significantly stimulated by chemical dehydration with 0.5 M sorbitol and 56% repetitive embryogenesis was achieved even after exposure to 2 M sorbitol for 24 h. The cotyledonary somatic embryos when alginate-encapsulated showed 47% repetitive embryogenesis even after chemical dehydration in 1.5 M sorbitol for 4 days followed by 1 h air dehydration, but failed to survive to the same dehydration conditions without encapsulation. The optimized repetitive embryogenesis and desiccation protocols offer the possibility to use in vitro techniques for continuous reliable somatic embryo production and short term germplasm storage.     

Nongraded Dehydration and Low-Pressure Infiltration for Rapid Celloidin Embedding of Brain Tissue

Various ways of shortening single steps in the celloidin process have been combined to form a routine method which may be completed, for tissues of average size, within a week following fixation. Fixed, washed tissue slices 5 mm thick are dehydrated in 1 or 2 changes of absolute ethanol and acetone, 1:1. This requires 24 hr in an incubator at 37 C, or 12-16 hr if a magnetic stirrer is used. After ether-alcohol for 4 hr. the tissues are transferred to 5% celloidin and infiltrated in a vacuum desiccator attached to a filter pump. When the volume of celloidin is reduced to half the original amount (about 2 hr), the tissues are removed from the infiltrating fluid and embedded in 10% celloidin. The blocks are hardened in chloroform and cleared by suspending them in 2 or 3 changes of terpineol agitated by a magnetic stirrer. Sections are cut in terpineol, using any type of microtome. After washing in 95% alcohol, they are mounted on albumenized slides for staining.     

An Improved Method for Rapid Fixation and Embedding of Pteridophyte Plant Materials

A rapid method for fixation and embedding of plant materials, especially pterido-phytes, is suggested. Addition of tannic acid following osmication improved the visualization of membranes. Staining en bloc with uranyl acetate between osmication and tannic acid is suggested for tissues infected with fungi and bacteria.     

A Quick Embedding Method for Light and Electron Microscopy of Textile Fibers

A quick embedding method employing UV polymerization reactions has been devised for embedding fibers in acrylic and meth-acrylate media. The resultant thin, flat embed-dings are suitable for both light and electron microscopy.     

A Quick Embedding Method for Light and Electron Microscopy of Textile Fibers

A quick embedding method employing UV polymerization reactions has been devised for embedding fibers in acrylic and meth-acrylate media. The resultant thin, flat embed-dings are suitable for both light and electron microscopy.     

A Plastic Embedding Technique for Analyzing Fluorescent Dextran-Amine Labelled Neuronal Profiles

A plastic embedding technique employing fluorescently labelled dextran-amines is described. After application of tracer to cut nerves and appropriate transport time, animals were fixed in paraformaldehyde. Subsequently their brains were dissected, heads and brains were dehydrated, embedded in methacrylate and sectioned serially on a rotary microtome. Plastic sections allow high resolution of single neuron profiles and complete serial reconstruction of un-distorted sections, including embryos with large amounts of yolk. In conjunction with whole mount analysis and double labelling, this technique can accurately reveal the spatial relationships of nerve components throughout development.     

A Method for Embedding Thin Membranes in Historesin

A method is described for flat-embedding thin membranous tissues in Historesin. It allows easy orientation for sectioning large areas parallel to the surface. Selected fields can be monitored from the unfixed specimen, throughout preparation, to mounting on the microscope slide. For cross-sectioning, the flat-embedded tissue can be stacked and re-embedded to increase the amount of material examined per section.     

Suitability of Different Protein A-Gold Markers for Immunogold-Silver Staining in Paraffin Sections

An incubation protocol to immunolabel Lowicryl semithin sections was applied to paraffin probes. To improve the labeling density, colloidal gold complexes of different preparations and sizes were compared. The type of colloidal gold preparation used was found to affect the specificity of the immunostaining. Gold colloid of 5 nm diameter particle size prepared with white phosphorus minimized nonspecific background labeling of β-casein in paraffin embedded sections of the mammary epithelium of pregnant mice. Gold colloids of 5 nm and 9 nm diameter particle size prepared in varying concentrations of tannic acid generated significant nonspecific staining in similar tissue preparations.     

Suitability of Different Protein A-Gold Markers for Immunogold-Silver Staining in Paraffin Sections

An incubation protocol to immunolabel Lowicryl semithin sections was applied to paraffin probes. To improve the labeling density, colloidal gold complexes of different preparations and sizes were compared. The type of colloidal gold preparation used was found to affect the specificity of the immunostaining. Gold colloid of 5 nm diameter particle size prepared with white phosphorus minimized nonspecific background labeling of β-casein in paraffin embedded sections of the mammary epithelium of pregnant mice. Gold colloids of 5 nm and 9 nm diameter particle size prepared in varying concentrations of tannic acid generated significant nonspecific staining in similar tissue preparations.     

Embedding Thin Plant Specimens for Oriented Sectioning

Small plant structures such as small primary roots, filamentous mosses and algae are difficult to orient for sectioning since they become wavy and curl during embedding. A method is described for embedding and orienting tiny plant specimens in a glycol methacrylate resin using self-constructed flat molds. Prior to sectioning, small samples can be oriented in both the longitudinal and the transverse plane. As several samples can be sectioned simultaneously, time-consuming trimming of the blocks is reduced substantially. The efficiency of this technique has been demonstrated using the tiny roots of the model plant Arabidopsis thaliana (L.) Heynh.     

The Hortega Silver Carbonate Method for Staining Pituicytes After Paraffin Embedding

In three papers published on pituicytes, of the ox by Bucy (1930), of the human by Shanklin (1940), and of the horse by Vazquez-Lopez (1942) the pituitaries were sectioned by the freezing method and stained by the Hortega silver carbonate technic. Since that time, as a routine procedure in our laboratory, frozen sections have been replaced by paraffin which in no way interferes with the Hortega silver carbonate staining.     

Replacing xylene with n-heptane for paraffin embedding

Abstract

In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.     

木本植物非均质化组织石蜡切片制作方法

在常规石蜡切片技术的基础上, 针对木本植物茎段木质化程度高、硬度大以及各部分组织硬度不均匀等特点, 选取核桃(Juglans regia)茎段以及芽接愈合区域组织为实验材料, 对固定、软化和脱水等关键步骤进行改进, 获得结构完整且染色清晰的茎段组织和嫁接愈合区域组织切片, 可清晰地观察到各部分组织的形态特征和愈合过程中的发育特征, 且缩短了制片周期。采用改良后的实验流程成功获得了苹果(Malus pumila)、桃(Amygdalus persica)、杏(Armeniaca vulgaris)、李(Prunus salicina)和杨(Populus tomentosa)的茎段横截面切片。该方法为从解剖学上研究林木茎段生长机制和形态发育变化提供技术基础, 为非均质化植物材料的石蜡切片制作提供参考。