A Simplified and Convenient Method for the Double-Embedding of Tissues

A method for embedding tissues with a celloidin-paraffin combination is presented. The essential features of the process depend upon (1) a thorough infiltration of the specimen with celloidin of low concentration, and (2) the subsequent impregnation of both the specimen and the celloidin with paraffin.

The methods for sectioning, and the removal of the embedding agent are given.

The chief advantages of this method are: the preservation of all of the advantages of celloidin embedding but with a great saving of time, and greater convenience of storage; the cutting of thin sections (2μ for many types of tissues); it is useful for embedding specimens for which neither pure paraffin nor pure celloidin are entirely satisfactory, i.e. those containing tissues differing in density.     

Oriented Embedding of Single-Cell Organisms

The dexribed technique facilitates oriented embedding of individual cells in various media for both light and electron microscopy. A fixed Specimen is embedded in a small cube of 2% agar at 40 C and subsequently sealed in the desired orientation to a strip of black paper which then serves as a tab for transferring the specimen during dehydrating and embedding procedures. The beveled ends of the strip indicate the exact location of the specimen in the cube. This technique can be employed for the embedding media used in both light and electron microscopy. It ah permits photomicrographs of the whole specimen to be made which can be compared with photomicrographs of individual sections cut from the specimen in a selected plane.     

8种不同方法保藏病原菌效果的对比观察

采用８种不同的菌种保藏方法,对１８种常见病原菌的保藏时间及生物学特性的影响进行了对比观察。结果表明：冷冻干燥法保藏菌种时间最长（本实验室已保存１５年）;肉浸汤琼脂平板法保藏菌种时间最短（２～３月）。保藏时间由长到短依次为：冷冻干燥法＞半固体冷冻法≥半固体斜面加液体石蜡覆盖法＞半固体斜面法＞肉浸汤加液体石蜡覆盖法＞血琼脂平板法＞肉浸汤法＞肉浸汤琼脂平板法。且相同方法对不同菌种保藏时间不同。保藏时间在１年以内的菌种,其生物学特性无明显变异;而经冷冻干燥法保藏时间较长的白喉棒状杆菌、金黄色葡萄球菌、甲型副伤寒沙     

Protective Paraffin Infiltration of Soft Tissues in Insects to Facilitate Softening of Hard Exoskeletons

This technique can produce serial sections as thin as 5 μ from hard chitin-covered materials of insects or other arthropods. Procedures: Fix with alcoholic Bouin's fluid for 3 hr. Henceforth subject material to partial vacuum in each step to ensure a final proper embedding. Wash with 80% ethanol 2 or 3 times for 2 hr or until the picric acid is largely removed. Dehydrate to 90% ethanol and give 2 changes of n-butanol 2 hr each, and one of a 1:1 n-butanol-paraffin mixture in 56-57° oven for 12 hr. Finally, use 2 baths of pure paraffin, 3 hr each, to complete the infiltration. After the last bath, withdraw the specimen from the paraffin, and remove the superficial paraffin, first mechanically and then with a xylene bath for 4 min. Rinse first with n-butanol, and afterwards with absolute ethanol, 2 min each. The compound eyes are protected with a paraffin covering, the specimen is hydrated with a 1% aqueous solution of detergent for 1 hr and then washed with running tap water. The material is treated with a concentrated sulfuric-nitric mixture (H₂SO₄:HNO₃) for 4 hr to eliminate the exoskeleton. After this treatment, the specimen is washed with running tap water for 12 hr, dehydrated with acetone and then bathed in a 2% solution of celloidin in ethyl acetate to form a protective artificial cuticle. This coating is hardened with 2 quick baths of chloroform, the specimen reembedded in paraffin, and the block cast for sectioning.     

Histological Techniques to Improve Testing of Pulpal Response of Teeth to Filling Material

Two fixation fluids, two fixation techniques and two embedding methods were investigated for their effects on the quality of sections of teeth for pulpal response to filling materials to improve evaluation of pulpal responses. Sections from 32 baboon teeth were prepared, half with experimental cavities and half without, using either 10% formaldehyde or 4% glutaraldehyde, longitudinal tooth splitting or removal of the tooth apex, and paraffin or K plast resin embedding; decalcification in a formic acid mixture was a constant throughout. Histometric analysis showed that paraffin embedding produced less shrinkage than the K Plast resin embedding although the difference was not statistically significant. Six parameters of separation at the pu1p:dentine interface were studied: embedding, fixative, presence or absence of a cavity, cutting technique and individual animal tooth type. Statistical investigation revealed that fixative, cutting technique, and fixative and cutting technique combined had significant influences on the separation artifact. Of the combinations tested the choice of embedding method depends on which of the two artifacts, shrinkage or separation, is more adverse in the opinion of the investigator. Four percent glutaraldehyde together with the longitudinal split technique of fixation. processed by either K Plast resin embedding or paraffin embedding produced satisfactory pulpal sections.     

Histological Techniques to Improve Testing of Pulpal Response of Teeth to Filling Material

Two fixation fluids, two fixation techniques and two embedding methods were investigated for their effects on the quality of sections of teeth for pulpal response to filling materials to improve evaluation of pulpal responses. Sections from 32 baboon teeth were prepared, half with experimental cavities and half without, using either 10% formaldehyde or 4% glutaraldehyde, longitudinal tooth splitting or removal of the tooth apex, and paraffin or K plast resin embedding; decalcification in a formic acid mixture was a constant throughout. Histometric analysis showed that paraffin embedding produced less shrinkage than the K Plast resin embedding although the difference was not statistically significant. Six parameters of separation at the pu1p:dentine interface were studied: embedding, fixative, presence or absence of a cavity, cutting technique and individual animal tooth type. Statistical investigation revealed that fixative, cutting technique, and fixative and cutting technique combined had significant influences on the separation artifact. Of the combinations tested the choice of embedding method depends on which of the two artifacts, shrinkage or separation, is more adverse in the opinion of the investigator. Four percent glutaraldehyde together with the longitudinal split technique of fixation. processed by either K Plast resin embedding or paraffin embedding produced satisfactory pulpal sections.     

ADaptation of A CRyostat QUenching APparatus to A PAraffin-LIke EMbedding TEchnique for IMproved FRozen SEctions

To eliminate cellular distortion and compression in a tissue block by the direct application of a heat extractor, such as found on a number of cryostats, a paraffin-like embedding method was developed for frozen tissue processing. For this technique, a heat extractor was adapted to fit flush against the back of the specimen plate as shown in figure 1 (for maximum heat removal) and the sides of the specimen plate were trimmed to permit placement on the paraffin embedding mold (Lab-Tek molds were used).     

A Method of Orienting Paraffin Sections for Three-Dimensional Reconstructions

To make reconstructions from serial sections, reference points are needed to orient the sections. Such points can be provided after paraffin embedding by cutting the bottom face of the block to form a plane and adding a groove along the center of this plane. The plane and groove are coated with Mimeograph Correction Fluid and the block is built up by dipping in hot paraffin so that the marked plane lies inside the block. Each section will have a blue line with a notch in it representing the plane and groove. This line remains through staining and is used to orient each section with respect to an eyepiece reticle. The reticle, in effect, supplies X and Y coordinates for every point in the specimen while the number of each section counted from one end is a Z coordinate.     

DNA提取的应用与相关技术分析

为了分析影响提取DNA的有关因素,采用标准酚—氯仿抽提法和蛋白酶消化法提取DNA。结果表明,平均每mL全血可提取200 ~ 300μgDNA;新鲜组织及OCT包埋冰冻组织提取DNA,平均每0.2g组织可获得200~300μgDNA;直接将石蜡标本提取物制作模版,PCR扩增良好。从4种组织中均获得较为纯净的DNA;OCT对组织DNA无不良影响。 Abstract:To explore influence factors of DNA extraction,the protease and phenol-chloroform method was used to extract DNA in whole blood,fresh tissues,frozen tissues embedding with OCT and tissues embedding in paraffin.It results that 200~300μg DNA was extracted from 1ml whole blood or 0.2g fresh tissues or frozen tissue embedding with OCT.DNA extracted from paraffin specimen can be directly used in PCR amplification.Purity DNA can be extracted from four kinds of different tissues.OCT hasn't harmful effect on tissue DNA extraction.     

ULTRASTRUCTURAL ANALYSIS OF HEMATOPOIETIC COLONIES DERIVED FROM HUMAN PERIPHERAL BLOOD : A Newly Developed Method

The ultrastructure of granulocyte colonies derived from normal human peripheral blood leukocytes cultured in semisolid media has been studied by a new method developed for this purpose. Fixation, dehydration, and embedding of the whole content of the Petri dish resulted in a block of Epon containing colonies made up of cells with the spatial orientation of those observed in living cultures. This permitted serial sectioning through entire colonies. Cell maturation in vitro appeared to parallel that of normal marrow. However, even the most mature cells retained cytoplasmic characteristics of more immature cells. This was particularly true for eosinophils which only rarely possessed granules with electron-dense crystalline "cores," a feature typical for mature eosinophils. In addition to the normal-appearing hematopoietic cells found within colonies, very large round or spindle-shaped cells were present between colonies and firmly attached to the bottom of the culture dish. Although the histochemical and functional characterization of these cells awaits further study, it is suggested that they are related to histiocytes or macrophages. The technique described here should prove valuable in studies of the development, differentiation, and interaction of many types of cells.     

Transmission acoustic microscopy of tissue sections (1 GHz)

Summary The acoustic microstructure of mouse small intestine has been studied with a transmission acoustic microscope working at 1 GHz and the influence of the histologic processing on the microacoustic pattern has been tested. Unstained thin sections provide pictures rich in details and highly contrasted. Gelatin has been used as hydrosoluble embedding medium and has been compared to paraffin. The former embedding procedure retained the viscoelastic properties of the specimen far more and provided the most detailed pictures. Osmiun tetroxide has been used to demonstrate acoustic staining.     

A Paraffin Method for Refractory Plant Materials

A modification of Zirkle's n-butyl alcohol method for dehydrating refractory plant material and embedding it in paraffin is described. The material is cut into small pieces and killed and fixed in CRAF III. It is dehydrated in Zirkle's n-butyl alcohol series, slightly modified. Infiltration is accomplished by adding chips of paraffin of low melting point (52°C.) to the bottle containing butyl alcohol and the tissues at a gradually increasing series of temperatures. The butyl-alcohol-paraffin mixture is gradually replaced by pure paraffin (melting point 56-58°C). The material is embedded in Fisher Tissuemat (56-58°C). Before microtoming, the block of embedded tissue is trimmed so that part of the specimen is exposed, and it is soaked in water until it cuts easily.     

Transmission acoustic microscopy of tissue sections (1 GHz). Histoacoustics and acoustic staining

The acoustic microstructure of mouse small intestine has been studied with a transmission acoustic microscope working at 1 GHz and the influence of the histologic processing on the microacoustic pattern has been tested. Unstained thin sections provide pictures rich in details and highly contrasted. Gelatin has been used as hydrosoluble embedding medium and has been compared to paraffin. The former embedding procedure retained the viscoelastic properties of the specimen far more and provided the most detailed pictures. Osmiun tetroxide has been used to demonstrate acoustic staining.     

Orienting Minute Objects for Sectioning in a Definite Plane

Minute objects can be prepared for sectioning in a definite plane by a method which reembeds them directly on the cutting block under a dissecting microscope. By melting the paraffin immediately around the specimen, the latter can be oriented with reference to the planes of the block. After trimming, the block can be oriented squarely with reference to the microtome knife. Objects as small as 0.2 mm. have been cut successfully. The material sectioned included carpel primordia of Lathyrus, and young embryos, shoot apices and young axillary buds of Pinus. The technic is simpler than most methods previously suggested and it permits good control over the plane of sectioning.     

Enhanced Histochemical Detection of Iron in Paraffin Sections of Mouse Central Nervous System Tissue: Application in the APP/PS1 Mouse Model of Alzheimer’s Disease

Histochemical methods of detecting iron in the rodent brain result mainly in the labeling of oligodendrocytes, but as all cells utilize iron, this observation suggests that much of the iron in the central nervous system goes undetected. Paraffin embedding of tissue is a standard procedure that is used to prepare sections for microscopic analysis. In the present study, we questioned whether we could modify the iron histochemical procedure to enable a greater detection of iron in paraffin sections. Indeed, various modifications led to the widespread labeling of iron in mouse brain tissue (for instance, labeling of neurons and neuropil). Sites of focal concentrations, such as cytoplasmic punctate or nucleolar staining, were also observed. The modified procedures were applied to paraffin sections of a mouse model (APP/PS1) of Alzheimer’s disease. Iron was revealed in the plaque core and rim. The plaque rim had a fibrillary or granular appearance, and it frequently contained iron-labeled cells. Further analysis indicated that the iron was tightly associated with the core of the plaque, but less so with the rim. In conclusion, modifications to the histochemical staining revealed new insights into the deposition of iron in the central nervous system. In theory, the approach should be transferrable to organs besides the brain and to other species, and the underlying principles should be incorporable into a variety of staining methods.     

Laboratory hints from the Literature: Department Devoted to Abstracts of books and papers from other Journals Dealing with stains and Microscopic Technic in General

A modification of Zirkle's n-butyl alcohol method for dehydrating refractory plant material and embedding it in paraffin is described. The material is cut into small pieces and killed and fixed in CRAF III. It is dehydrated in Zirkle's n-butyl alcohol series, slightly modified. Infiltration is accomplished by adding chips of paraffin of low melting point (52°C.) to the bottle containing butyl alcohol and the tissues at a gradually increasing series of temperatures. The butyl-alcohol-paraffin mixture is gradually replaced by pure paraffin (melting point 56-58°C). The material is embedded in Fisher Tissuemat (56-58°C). Before microtoming, the block of embedded tissue is trimmed so that part of the specimen is exposed, and it is soaked in water until it cuts easily.     

Histological investigation of organisms with hard skeletons: a case study of siliceous sponges.

Siliceous and calcareous sponges commonly are treated with acid to remove the spicules prior to embedding and cutting for histological investigations. Histology of spiculated sponge tissue represents a challenging problem in sponge histotechnology. Furthermore, fluorescence in situ hybridization (FISH), a key method for studying sponge-associated microbes, is not possible after acid treatment. For a broad range of siliceous sponge species, we developed and evaluated methods for embedding in paraffin, methylmethacrylate resins, LR White resin and cryomatrix. Different methods for cutting tissue blocks as well as mounting and staining sections also were tested. Our aim was to enable histological investigations and FISH without prior removal of the spicules. To obtain an overview of tissue and skeleton arrangement, we recommend embedding tissue blocks with LR White resin combined with en bloc staining techniques for large specimens with thick and numerous spicules, but paraffin embedding and subsequent staining for whole small specimens. For FISH on siliceous sponges, we recommend Histocryl embedding if the spicule content is high, but paraffin embedding if it is low. Classical histological techniques are used for detailed tissue examinations.     

Histological investigation of organisms with hard skeletons: a case study of siliceous sponges

Siliceous and calcareous sponges commonly are treated with acid to remove the spicules prior to embedding and cutting for histological investigations. Histology of spiculated sponge tissue represents a challenging problem in sponge histotechnology. Furthermore, fluorescence in situ hybridization (FISH), a key method for studying sponge-associated microbes, is not possible after acid treatment. For a broad range of siliceous sponge species, we developed and evaluated methods for embedding in paraffin, methylmethacrylate resins, LR White resin and cryomatrix. Different methods for cutting tissue blocks as well as mounting and staining sections also were tested. Our aim was to enable histological investigations and FISH without prior removal of the spicules. To obtain an overview of tissue and skeleton arrangement, we recommend embedding tissue blocks with LR White resin combined with en bloc staining techniques for large specimens with thick and numerous spicules, but paraffin embedding and subsequent staining for whole small specimens. For FISH on siliceous sponges, we recommend Histocryl embedding if the spicule content is high, but paraffin embedding if it is low. Classical histological techniques are used for detailed tissue examinations.     

A Simple Method for Paraffin and Plastic Embedding of Protozoa

SUMMARY. To avoid multiple centrifugation and considerable losses of material in preparing protozoa for paraffin and plastic embedding a simple method has been worked out, requiring only two centrifugations, one before and one after fixation. During the second centrifugation, which is done at 700 g for 10 minutes, the organisms form a cohesive pellet which may be removed from the centrifuge tube by means of a fine spatula and handled as a piece of tissue through the whole process of dehydration and embedding.     

桦树松萝的石蜡切片方法改良及形态学研究

以传统石蜡切片方法为基础,在固定、软化、包埋、染色等具体方法上进行了适合松萝属植物特点的改良。结果表明:采用改良制片方法获得了染色清晰、组织完整的桦树松萝切片。桦树松萝地衣体横切面结构从外到内依次划分为皮层、藻层、髓层和中轴;皮层是由横向分裂的菌丝交织成类似于高等植物的假厚壁组织,藻层由大量共生藻的藻细胞和菌丝所组成;髓层由疏松的菌丝体组成;中轴由致密的菌丝体组成。