Piccolyte Investments for Better Section Cutting

Piccolyte 115 (β-pinene polymers) added to Tissuemat, Paraplast or Peel-Away embedding media is recommended for investment of paraffin infiltrated tissues. Mixed with paraffin at 3% and 10% and used for double embedding of paraffin infiltrated tissues, Piccolyte 115 permits good, complete sections virtually free of folds or wrinkles in less time and with less effort than with paraffin embedding alone.     

A Rapid Rosin-Celloidin-Paraffin method for Embedding Tissues

Pieces of tissue of various sizes or tissue fragments are dehydrated in 95% alcohol, cleared, washed with ether and infiltrated with a solution containing parloidin 9.6 g., camphor 3.0 g., absolute alcohol 200.0 ml., ether 200.0 ml., rosin 45.0 g. and castor oil 10 drops. After evaporation to the desired consistency, the mass is hardened with chloroform vapor, trimmed, passed through 3 changes of xylene to remove the rosin, embedded in paraffin, sectioned serially, stretched and stained as for paraffin methods. Methods of defatting tissues and detailed procedure for embedding fragments of bone marrow by this method are given.     

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.     

Combined Carbowax-Paraffin Technic for Microsectioning of Fixed Tissues

A combined Carbowax-paraffin technic for microsectioning fixed tissues gave ribbon sections as do paraffin infiltrated and embedded tissues. Blocks of formalin or alcohol fixed tissues 2 mm. thick were infiltrated with H.E.M. (Polyethlene Glycol: Carbowax, Hartman-Leddon Co.) or with one of the following polyethylene glycol ester waxes (Glycol Products Co., Inc.) for 4 hours at (61°C.): Polyethylene Glycol 600(Di) Stearate; Carbowax 1000-(Mono) Stearate; Carbowax 4000 (Mono) Stearate; Carbowax 4000 (Mono) Laurate; Carbowax 6000 (Mono) Oleate. The Carbowax infiltrated tissues were placed for 10 minutes in xylene (61° C.), into paraffin (61° C.) for 30 minutes, then into molten paraffin contained in separate molds. (The xylene passage can be excluded for preparations which preclude its use). The blocks were hardened rapidly by submerging in ice water and were fastened to carriers as in the usual paraffin technic. Tissues were cut 6 µ thick. Segments of ribbon were spread on a water bath and mounted on slides. After drying, tissues were stained directly with hematoxylin-eosin or were carried through xylene and alcohols as in routine paraffin preparations prior to staining. The Sudan III fat stain and Best's carmine stain for glycogen were applied as in usual technics. Cellular detail was well preserved and structures did not show the extent of distortion and shrinkage encountered in ordinary paraffin technic preparations.     

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.     

The application of polyethylene glycol (PEG) to electron microscopy

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine- coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis.     

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.     

Hematoxylin Toluidine Blue-Phloxinate Staining of Glycol Methacrylate Sections of Retina and Other Tissues

For a detailed study of the developing chick retina a technique has been developed using glycol methacrylate embedding and a hematoxylin toluidine blue-phloxinate stain. After removal of the vitreous body, one half-segment of the eye is dehydrated through graded ethyl alcohols to 95%, infiltrated and embedded in glycol methacrylate, and sectioned at 2 μm. The sections are stained in alum hematoxylin and then in a mixture containing toluidine blue-phloxinate from a stock solution of the dye that has matured for 2-3 weeks. Differentiation is not required and there is only slight staining of the plastic matrix. The quality and clarity of the sections contrasts markedly with that of similarly stained 5 μm paraffin wax sections of the retina. This technique has also been applied to skin, spinal cord, dorsal root ganglia, pancreas and small intestine. The stained sections from these tissues have proved very useful in revealing structural components.     

Histology of plastic embedded amphibian embryos and larvae

Amphibians including the South African clawed frog Xenopus laevis, its close relative Xenopus tropicalis, and the Mexican axolotl (Ambystoma mexicanum) are important vertebrate models for cell biology, development, and regeneration. For the analysis of embryos and larva with altered gene expression in gain-of-function or loss-of-function studies histology is increasingly important. Here, we discuss plastic or resin embedding of embryos as valuable alternatives to conventional paraffin embedding. For example, microwave-assisted tissue processing, combined with embedding in the glycol methacrylate Technovit 7100, is a fast, simple, and reliable method to obtain state-of-the-art histology with high resolution of cellular details in less than a day. Microwave-processed samples embedded in Epon 812 are also useful for transmission electron microscopy. Finally, Technovit-embedded samples are well suited for serial section analysis of embryos labeled either by whole-mount immunofluorescence, or with tracers such as GFP or fluorescent dextrans. Therefore, plastic embedding offers a versatile alternative to paraffin embedding for routine histology and immunocytochemistry of amphibian embryos.     

A Tissue Basket for Paraffin Infiltration

The processes of washing, dehydration and paraffin infiltration when large numbers of tissues must be individually identified are always laborious. Washing and dehydration can be carried out in individual glass vials with relative ease and fairly rapidly if gravity flow reagent bottles are used to fill the vials. The use of similar vials for paraffin infiltration is usually complicated by hardening of the paraffin if too many vials are removed from the oven at once, or by cooling of the oven itself if it is too frequently opened. The same criticism applies to the process of embedding tissues when large numbers of vials must be handled simultaneously.     

Pasternack's Paraffin Method Modified for Plant Tissue

This method for preparing paraffin sections of plant material is a modification of Pasternack's one-hour method for animal tissues. Fixation in Randolph's CRAF fixative is hastened by heat and increased vapor pressure obtained by the use of screw top vials. Dehydration with Zirkle's butyl alcohol series likewise is hastened in the same manner. The rapid penetration of paraffin by the use of 1/2 paraffin and 1/2 butyl alcohol in heated screw top vials shortens the embedding process. Sections are held on the slide thru staining by albumen fixative and a coating of 0.2% celloidin in absolute alcohol and ether. Good penetration with freedom from shrinkage or distortion is obtained and root tip chromosome counts can be made in approximately 3 hours.     

A Tissue Basket for Paraffin Infiltration

The processes of washing, dehydration and paraffin infiltration when large numbers of tissues must be individually identified are always laborious. Washing and dehydration can be carried out in individual glass vials with relative ease and fairly rapidly if gravity flow reagent bottles are used to fill the vials. The use of similar vials for paraffin infiltration is usually complicated by hardening of the paraffin if too many vials are removed from the oven at once, or by cooling of the oven itself if it is too frequently opened. The same criticism applies to the process of embedding tissues when large numbers of vials must be handled simultaneously.     

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.     

A Celloidin Infiltration-Frozen Section Sequence for Enhanced Preservation of Phosphatases in Bone

The effect of the following embedding procedures on the acid and alkaline phosphatase content of decalcified mouse tibiae has been studied: embedding in 23% gelatine for 18 hr at 37° C, embedding in paraffin wax in vacuo for 1 hr at 58° C, and impregnation with 4% celloidin in diethyl ether and ethanol at 4° C for 2-3 days. Unsupported tissues were also used to demonstrate these enzymes for comparison with the above procedures. Tibiae were first fixed in 10% neutral formalin at 4° C for 15 hr, decalcified in equal volumes of 2% formic acid and 20% sodium citrate at pH 4.9 for not more than 5 days and then washed in distilled water before carrying out the embedding schedules. The celloidin-impregnated tibiae were placed in 70% ethanol to harden the celloidin and then washed in distilled water for 1-2 hr. These tibiae and those embedded in gelatine were cast in a gelatine block which was then hardened in 10% neutral formalin at 4° C for 2 hr. Sections of these and unsupported tibiae were cut at 15 μ on a freezing microtome. Decalcified tibiae embedded and blocked in paraffin wax were sectioned at 15 μ on a base sledge microtome. The enzymes were demonstrated using the coupling azo dye method given by Pearse (Histochemistry, 1st Ed. 1954). The stable diazotates of 4 benzoyl amino 2-5 diethoxyanilene, 3 nitro toluidine and o-dianisidine were used. Of the embedding procedures paraffin wax embedding produced the greatest loss of both enzymes. Gelatine embedding and infiltration with celloidin were equally good for the demonstration of acid phosphatase but for alkaline phosphatase the celloidin method was superior. The gelatine embedded material did not produce consistently good results. Celloidin-impregnated tibiae could be stored without marked deterioration of the enzyme content for longer than gelatine-embedded tibiae.     

Freeze drying and freeze substitution combined with low temperature-embedding

Summary An X-ray microanalytical and morphological investigation was carried out on rapidly frozen freeze-dried or freeze-substituted tissues. A comparison was made between different embedding and polymerisation procedures following freeze substitution and freeze drying. The investigation also included an analysis of specimens which had been infiltrated, embedded and polymerised by ultraviolet irradiation at low temperatures with Lowicryl-HM20. The method of freeze drying, followed by embedding and polymerisation at low temperatures in vacuo was found to give satisfactory results, comparable with more tedious and hazardous freeze substitution technique.     

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.     

Stain for histamine in mast cells in fixed tissue

Summary A technique is described for staining histamine in mast cells of tissues fixed in alcohol. The method employs cold ethanol fixation, xylene clearing, paraffin embedding, and staining of the sections with 1% o-phthalaldehyde in ethyl benzene in a humid chamber. This study was supported by a grant from the John A. Hartford Foundation.     

The use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding.

Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or tricholoracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green-pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.     

A Device for Orienting and Embedding Minute Objects

A device is described for embedding objects 200μ or less in paraffin in accurate orientation. It consists of a small paraffin bath heated by a removable coil of wire, and having a water jacket for rapid cooling of the block. Orientation is effected under the microscope using electrically heated needles.