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1.
The preservation of many stained gross specimens in solution creates some difficulties. It is convenient and effective to preserve material in polyester resin instead of glycerol. The aim of this study was to determine the usefulness of clearing and embedding using polyester resin. The samples consisted of the nerve distribution patterns of skeletal muscles stained using Sihler's method. The muscles were cleared more successfully and the intramuscular nerve distributions were demonstrated better in polyester than in glycerol. The method presented here eliminates not only the storage and handling problems of specimens, but also problems such as pale stains and the molding of preparations. Furthermore, it is more convenient to examine and to photograph specimens cleared and embedded in polyester than those stored in glycerol. 相似文献
2.
The preservation of many stained gross specimens in solution creates some difficulties. It is convenient and effective to preserve material in polyester resin instead of glycerol. The aim of this study was to determine the usefulness of clearing and embedding using polyester resin. The samples consisted of the nerve distribution patterns of skeletal muscles stained using Sihler's method. The muscles were cleared more successfully and the intramuscular nerve distributions were demonstrated better in polyester than in glycerol. The method presented here eliminates not only the storage and handling problems of specimens, but also problems such as pale stains and the molding of preparations. Furthermore, it is more convenient to examine and to photograph specimens cleared and embedded in polyester than those stored in glycerol. 相似文献
3.
Daniel Chappard Christian Alexandre Sabine Palle Georges Riffat 《Biotechnic & histochemistry》1986,61(3):145-149
A one-step clearing and embedding procedure for alizarin red S stained skeletons is described. Embryos are fixed in formalin, skinned and eviscerated. After staining in a 10 mg/liter solution of alizarin red S in 5% aqueous KOH, specimens are dehydrated in a graded series of acetone-polyester monomer solutions. Finally, the specimens are embedded at room temperature in the polyester resin. A special reusable metallic mold is described for embedment of large fetuses. Specimens previously cleared in glycerol can be processed with this method. 相似文献
4.
A one-step clearing and embedding procedure for alizarin red S stained skeletons is described. Embryos are fixed in formalin, skinned and eviscerated. After staining in a 10 mg/liter solution of alizarin red S in 5% aqueous KOH, specimens are dehydrated in a graded series of acetone-polyester monomer solutions. Finally, the specimens are embedded at room temperature in the polyester resin. A special reusable metallic mold is described for embedment of large fetuses. Specimens previously cleared in glycerol can be processed with this method. 相似文献
5.
C G Rousseaux 《Stain technology》1985,60(5):295-297
An automated, rapid procedure for differential staining of cartilage and bone of vertebrates is described. The process involves rapid, complete staining of freshly skinned, eviscerated specimens after 30 sec immersion in a 70 C water bath, fixation in formol acetic alcohol and a rinse in 70% alcohol. Using an automatic tissue processor, the specimen is stained in alcian blue for 24 hr and macerated in 3% potassium hydroxide for 8 hr. Staining in alizarin red with maceration in 3% potassium hydroxide is completed manually. The specimens are cleared and preserved in glycerol. Good quality evenly stained specimens can be examined in less than three days and up to 600 fetuses can be processed in less than five days. 相似文献
6.
C. G. Rousseaux 《Biotechnic & histochemistry》1985,60(5):295-297
An automated, rapid procedure for differential staining of cartilage and bone of vertebrates is described. The process involves rapid, complete staining of freshly skinned, eviscerated specimens after 30 sec immersion in a 70 C water bath, fixation in formol acetic alcohol and a rinse in 70% alcohol. Using an automatic tissue processor, the specimen is stained in alcian blue for 24 hr and macerated in 3% potassium hydroxide for 8 hr. Staining in alizarin red with maceration in 3% potassium hydroxide is completed manually. The specimens are cleared and preserved in glycerol. Good quality evenly stained specimens can be examined in less than three days and up to 600 fetuses can be processed in less than five days. 相似文献
7.
Cleared and stained specimens may become faded and/or opaque after long periods in preservative solutions. We developed a technique to revitalize faded and/or destained specimens regardless of their age. By adapting and revising Wassersug's procedure for differential staining of bone and cartilage, we successfully cleared and restained numerous small amphibian specimens of various ages. After rehydrating, specimens were bleached in potassium hydroxide and hydrogen peroxide, stained with alcian blue, and macerated with trypsin as needed. Alizarin red stain was applied or reapplied, although staining occasionally was unsuccessful in old formalin fixed specimens. After restaining, the specimens were dehydrated and placed in glycerol. 相似文献
8.
Various schedules for staining fetal rat skeleton with alizarin red S were tested to determine a procedure that would produce a completely cleared and well-stained specimen in a short period of time. A 2 day procedure is presented which can produce specimens that are satisfactory but not completely transparent. A 7 day procedure produces cleared and stained specimens which can be well visualized with a dissecting microscope (30×). Fetal rats of 21 days gestation were fixed in 10% formalin for at least 1 wk. The specimens were skinned and eviscerated and then dehydrated in 2 changes of acetone for 12 hr (8 ml per gram body weight). The specimens were then placed in 1% KOM-alizarin red S (6 mg/liter) or 3 days, followed by 10% KOH-alizarin red S for 3 days. Finally, the specimens were placed in a mixture of benzyl alcohol, ethanol, and glycerol (1:2:2) (4 ml per gram body weight) for 12 hr, and then transferred to pure glycerol for storage. 相似文献
9.
Differential skeletal staining is an important part of developmental toxicologic studies. Traditionally these studies have required time-consuming differentiation of one or both stains used and careful attention to the maceration step to prevent specimen destruction. We present a fully automated protocol which does not require differentiation of either dye and incorporates a controlled maceration step which is highly reproducible. This has resulted in high quality staining that is reproducible, stable, and can be done in volume with minimal personnel time. The process involves the staining of skinned, eviscerated specimens fixed in 95% ethanol. Using an automated tissue processor, the specimen is stained in alcian blue for 24 hr, macerated in 3% potassium hydroxide for 24 hr and stained with murexide for 24 hr. The specimens are cleared and preserved in glycerol. Within three days specimens have red stained bone and blue stained cartilage. The procedure was developed using 20-day-old Sprague-Dawley rat fetuses to evaluate the feasibility of using the procedure for teratology studies involving the fetal skeleton. Evenly stained specimens can be examined within three days and stored for years without loss of staining. 相似文献
10.
《Biotechnic & histochemistry》2013,88(4):193-196
Cleared and stained specimens may become faded and/or opaque after long periods in preservative solutions. We developed a technique to revitalize faded and/or destained specimens regardless of their age. By adapting and revising Wassersug's procedure for differential staining of bone and cartilage, we successfully cleared and restained numerous small amphibian specimens of various ages. After rehydrating, specimens were bleached in potassium hydroxide and hydrogen peroxide, stained with alcian blue, and macerated with trypsin as needed. Alizarin red stain was applied or reapplied, although staining occasionally was unsuccessful in old formalin fixed specimens. After restaining, the specimens were dehydrated and placed in glycerol. 相似文献
11.
Historically, some fetuses for regulatory developmental toxicity studies have been stained with alizarin red S and cleared with glycerol to visualize the ossified portion of their skeletons. Interest in examining cartilage arose owing to its inclusion in some regulatory guidelines. Methods for double staining rat skeletons have been published previously. The method described here for staining mouse skeletons is fully automated and uses alizarin red S to stain bone and Alcian blue to stain cartilage. Pregnant mice (Crl:CD1) were euthanized on gestation day 18 to obtain fetal specimens. Day 0 post-partum mouse pups also were stained. Our method was developed using the Shandon Pathcentre , which is a fully enclosed automated staining system that allows staining to be carried out at 30° C with a final clearing at 35° C. Our method uses the same solutions as for fetal rat processing, but with reduced time periods for the smaller size of mice vs. rat specimens. Staining, maceration and clearing of the specimens requires approximately 2 days. The time required of laboratory personnel, however, is minimal, because all solutions are changed automatically and the specimens do not require examination or removal from the processor until processing is complete. After processing, the specimens are suitable for immediate assessment of bone and cartilage. A mouse developmental toxicity study using 20 animals/group and approximately 10 fetuses/animal could be processed in only three runs using one machine. 相似文献
12.
J K Hartsfield L B Holmes 《Journal of craniofacial genetics and developmental biology》1988,8(4):345-350
Two methods of measuring cranial structures, the direct method and a photographic method, have been compared in day 18 mouse fetuses that have been stained with alizarin red S and cleared. Variation was determined for repeated measurements with each technique of the same specimen as well as the variation among specimens. There was variation in the results of direct measurements of the same specimens, but this was significantly (P less than 0.05) less than the variation among specimens, and the variation of measurements of the same specimen made using the photographic method was not significantly (P greater than 0.2) different from the variation among the specimens, and the variation of the specimens was not significantly (P greater than 0.2) different between the photographic and direct methods. Direct measurement is recommended for making linear measurements on the cleared skull. The photograph method allows better delineation of angles and calculation of surface area. 相似文献
13.
The usefulness of alkaline solutions for clearing the uterus and staining implantation sites in rats
The alkaline solution method for detecting uterine implantation sites (metrial glands or spots after delivery), which indicate the position of the embryo, was compared with conventional methods using Turnbull blue (Salewski's technique) and benzyl benzoate (Orsini's technique) in rats. When rat uteri were immersed directly in 2% sodium hydroxide [NaOH], potassium hydroxide [KOH] solution or one of several other alkaline solutions for one hour, the uteri were cleared. Then the metrial glands could be observed without further staining, and the spots after delivery with further staining. After alkaline treatment, unstained metrial glands appeared as yellowish-white degenerated masses and stained spots after delivery appeared as yellowish-brown patches covered with yellowish-white lumps. These yellowish-brown or yellowish-white sites were probably stained by the iron in hemosiderin deposits in the placental scar or in a vestige of the metrial glands left over from pregnancy. Formalin-fixed uteri were not cleared by immersion in a high concentration of NaOH, KOH or other alkaline solutions, but the spots after delivery were stained by this treatment. Turnbull blue did not stain the metrial glands, which contain no iron. Benzyl benzoate cleared fixed or unfixed uteri and allowed observation of the metrial glands and spots after delivery although the entire process required a few days and the specimens became hard and shriveled. The best and most convenient solution for clearing the rat uterus and for staining implantation sites was 2% NaOH or KOH. 相似文献
14.
The method of tissue embedding in melamine resin was applied to rat skeletal muscle. This method does not require tissue dehydration with organic solvents; only aqueous solutions are used. Electron micrographs of muscles embedded in melamine differ from those embedded in the conventional epoxy resin. In melamine-embedded muscles the actin and myosin filaments appear larger in diameter and subunits can be recognized in cross-sectioned myosin filaments. Within the Z-line, the characteristic patterns described for muscles embedded in epoxy resin are not visible; the spaces between the actin filaments are filled with electron-dense material. This suggests that the Z-line is more compact than could be concluded from epoxy resin-embedded muscle specimens. The M-line appears to be different from what is observed in epoxy-embedded muscle. The membranes appear as several clearly delineated layers. Dehydration rather than the action of the organic solvents per se is the main reason for the differences in the structure of the contractile apparatus between melamine- and epoxy-embedded muscles. 相似文献
15.
A technique which should be generally applicable for preparing permanent mounts of tissue cleared in Herr's four-and-a-half clearing fluid is described. This technique involves transferring plant or animal tissues through a series of solutions consisting of Pienarr's fixative, Herr's clearing fluid, chloral hydrate, acetone and finally polyester resin for mounting. Material prepared using this method is exceptionally transparent and well preserved, and is suitable for either phase contrast or Nomarski interference microscopy. 相似文献
16.
Sections of undemineralized bone embedded in a polyester resin and cut at 6 μ are stained for 10 min, without removal of the embedding matrix, in an aqueous solution composed of Solochrome cyanine R, 1 gin; glacial acetic acid, 2 ml; and distilled water, 98 ml. A pH about 2 is obtained by the acetic acid. The sections are washed and differentiated in tap water at 30 C, dehydrated in ascending alcohols, cleared and mounted in synthetic resin. “Young osteoid” stains light orange and, in the rest of an osteoid seam, two types of lamellae can be distinguished: one blue layer of ground substance or collagen and one orange layer of fibrillar collagen. The “calcification front” is sharply demarcated by its dark blue color. 相似文献
17.
G. R. Bullock M. K. Jasani J. M. Roberts I. H. M. Williamson 《The Histochemical journal》1985,17(2):143-151
Summary Tissues embedded in resin are convenient for routine use when the presence or absence of fibrin in them is to be confirmed using the electron microscope. To visualize fibrin using the light microscope, sections (1.0–2.0 m) from such specimens should be stained with Methylene Blue-Azure II-Basic Fuchsin (MBBF). Staining with MBBF is more controllable than with other methods and it requires only two short staining steps. Compared with Giemsa, MBBF provides a polychromatic, as opposed to a monochromatic end-result, sharply contrasting fibrin (blue) against collagen (pink-violet). 相似文献
18.
《Biotechnic & histochemistry》2013,88(2):125-128
A technique which should be generally applicable for preparing permanent mounts of tissue cleared in Herr's four-and-a-half clearing fluid is described. This technique involves transferring plant or animal tissues through a series of solutions consisting of Pienarr's fixative, Herr's clearing fluid, chloral hydrate, acetone and finally polyester resin for mounting. Material prepared using this method is exceptionally transparent and well preserved, and is suitable for either phase contrast or Nomarski interference microscopy. 相似文献
19.
20.
C. Knabe B. Kraska C. Koch U. Gross H. Zreiqat M. Stiller 《Biotechnic & histochemistry》2006,81(1):31-39
To evaluate the osteogenic potential of novel implant materials, it is important to examine their effect on osteoblastic differentiation. Characterizing the tissue response at the bone-biomaterial interface in vivo at a molecular level would contribute significantly to enhancing our understanding of tissue integration of endosseous implant materials. We describe here a new technique that overcomes difficulties commonly associated with performing immunohistochemistry on undecalcified sawed sections of bone. Sheep mandible specimens were fixed in an ethanol based fixative to maintain adequate antigenicity of the tissue. As a result, it was possible to omit antigen retrieval at high temperature for recovery of antigenicity, and detachment of sections from the slides was avoided. Following dehydration and infiltration, the specimens were embedded in a resin composed of polymethylmethacrylate and polybutylmethacrylate. Polymerization was achieved by adding benzoylperoxide and N,N-dimethyl-toluidine. This resin was selected because it maintained the antigenicity of the tissue, provided adequate properties for cutting 50 µm thick sections, and it facilitated deacrylizing the sawed sections. Acid-resistant acrylic slides were glued to the blocks using an epoxy resin based two-component adhesive to avoid detachment of the slides during the deacrylation procedure. Samples were stained for alkaline phosphatase, type I collagen, osteonectin, osteopontin, osteocalcin and bone sialoprotein. The EnVision + ™ dextran polymer conjugate two-step visualization system was applied for immunohistochemical detection of these bone matrix proteins. This procedure yielded positive staining for the osteogenic markers in cells and matrix components. The protocol described here facilitates the use of immunohistochemistry on resin embedded sawed sections of bone and provides a convenient and reliable method that can be used routinely for immunohistochemical analysis of hard tissue specimens containing implant materials. 相似文献