A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals

A simple, rapid procedure for dual staining of cartilage and bone in rodents, particularly in late gestation, has been developed for routine use. The procedure involves rapid, complete skinning of fresh eviscerated specimens following a 30 sec immersion in a 70 C water bath. The unfixed specimen is stained in a mixture of 0.14% Alcian blue and 0.12% alizarin red S in ethanol and glacial acetic acid. Specimens are then macerated in 2% KOH, cleared and hardened in 1:1 glycerin and distilled water, and stored in pure glycerin. Rapid staining of cartilage only is done in a mixture of 0.08% Alcian blue, glacial acetic acid, and ethanol, with subsequent maceration, clearing, and hardening as in the double staining procedure. Rapid staining of bone only, concurrent with maceration of soft tissue, can be done by placing fresh, unskinned specimens in a diluted mixture of alizarin red S in 2% KOH, with subsequent clearing and hardening in 1:1 distilled water and glycerin. Good quality fetal specimens can be prepared for examination by any of these procedures in a minimum of 11/2-2 days as compared to a minimum of 4-5 days for other procedures. Double stained specimens can be examined for abnormalities of the cartilage as well as bone.     

An automated technique for double staining mouse fetal and neonatal skeletal specimens to differentiate bone and cartilage

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.     

Alizarin Red S and Toluidine Blue for Differentiating Adult or Embryonic Bone and Cartilage

This technic has been successfully employed by the author for staining, in toto, the bones and cartilage of mature specimens of Urodela and the developing bone and cartilage of the embryonic human, cat, pig and rat. The differential staining is accomplished by using a modification of Dawson's method of staining bone with alizarin red S following a toluidine blue solution specific for cartilage. Specimens are fixed in 10% formalin, stained one week in a solution of .25 g. of toluidine blue in 100 cc. of 70% alcohol, macerated 5 to 7 days in a 2% KOH solution, counterstained for 24 hours in a 0.001% solution of alizarin red S in 2% aqueous KOH, dehydrated in cellosolve and cleared in methyl salicylate. In the adult and embryonic forms thus treated the soft tissues are cleared while the osseous tissue is stained red, the cartilage blue.     

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification

Abstract

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.     

A procedure for differential staining of cartilage and bone in whole formalin-fixed vertebrates.

This paper describes a modification of the Simons and Van Horn (1971) procedure for rendering cartilage blue, bone red, and soft tissue translucent or transparent in whole vertebrate specimens. Alcian blue and alizarin red S are used to stain cartilage and bone respectively. In our procedure formalin is used as a fixative. This is a significant modification because formalin is the common fixative for museum specimens. This clearing and staining procedure is thus readily applicable to comparative studies in anatomy, embryology and systematic zoology.     

A two-color acid-free cartilage and bone stain for zebrafish larvae

Traditionally, cartilage is stained by alcian blue using acidic conditions to differentiate tissue staining. The acidic conditions are problematic when one wishes to stain the same specimen for mineralized bone with alizarin red, because acid demineralizes bone, which negatively affects bone staining. We have developed an acid-free method to stain cartilage and bone simultaneously in zebrafish larvae. This method has the additional advantage that PCR genotyping of stained specimens is possible.     

Detection of Mineralized Structures in Early Stages of Development of Marine Teleostei Using a Modified Alcian Blue-Alizarin Red Double Staining Technique for Bone and Cartilage

We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100-200 ixm in length can be clearly visualized. The method is suitable for both onto-genic studies during early stages of skeletal development in most marine fishes (e.g., Sporus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 100% ethanol for up to two years.     

Simultaneous Differential Staining of Cartilage and Bone in Rodent Fetuses: an Alcian Blue and Alizarin Red S Procedure without Glacial Acetic Acid

Differential staining of cartilage and bone has several applications including developmental toxicology studies of new chemical candidates for pharmaceutical, industrial, and environmental use. It has been more common to stain fetal bone only using the dye alizarin red S: however, failure to evaluate the cartilaginous portion of the skeleton may result in the failure to identify toxicologically important alterations in skeletal morphology. Previously, differential staining of fetal cartilage and bone was best achieved by combining alizarin red S for staining bone with alcian blue to stain cartilage in glacial acetic acid solution: however, occupational hazards posed by the use of glacial acetic acid make these methods undesirable. Replacement of the glacial acetic acid with potassium hydrogen phthalate eliminates these hazards without compromising the quality of the stained specimen.     

Isolation and characterization of osteogenic cells derived from first bone of the embryonic tibia

Osteogenesis in the embryonic long bone rudiment occurs initially within an outer periosteal membrane and subsequently inside the cartilaginous core as a consequence of the endochondral ossification process. In order to investigate the development of these two different mechanisms of bone formation, embryonic chick tibial cell isolates were prepared from sites of first periosteal bone formation and from the immediately underlying hypertrophic cartilaginous core region. Mid-diaphyseal periosteal collars and the corresponding cartilage core were microdissected free from Hamburger-Hamilton stage 35 (Day 9) chick tibias and separately digested with a trypsin-collagenase enzyme mixture. The released cell populations were cultivated in vitro and characterized by morphological analysis, histochemical localization of alkaline phosphatase, alizarin red S staining for mineral deposition, growth rate [( 3H]thymidine uptake), and proteoglycan content. Results of these studies showed that periosteal collar cell cultures form nodule-like structures that stain positive with alkaline phosphatase and alizarin red S. Light and electron microscopic observation revealed cell and matrix morphologies similar to that of intact periosteum. The nodules were composed of plump cell types embedded within a mineralized matrix surrounded by a fibroblastic cell layer. Core cartilage cell cultures displayed typical characteristics of the hypertrophic state in their visual appearance and proteoglycan composition. The formation of osseous-like structures in periosteal collar cell cultures but not in core chondrocyte cell cultures demonstrates the relatively autonomous nature of intramembranous ossification while emphasizing the dependence of the endochondral ossification process upon an intact vascularized environment present in the developing tibia.     

Selective staining methods for cartilage of rat fetal specimens previously treated with alizarin red S.

A convenient method for staining cartilage with several basic stains after alizarin red S staining of bone was investigated in rat fetuses. It was found that bromophenol blue was useful and effective for staining of the margin and center areas of cartilage, even in specimens stored in glycerin for over 10 years. The specimens were washed in running tap water for 1 hr, and subsequently were immersed in water or in 70% ethanol at pH 4 for 1 hr or longer. The specimens were then stained with 0.005% bromophenol blue in 40% ethanol adjusted to pH 4 for 2 hr, or with 0.001% bromophenol blue in 40% ethanol adjusted to pH 4 for 24 hr. Furthermore, the bromophenol blue stain color actually faded when the specimens were immersed in water or in 70% ethanol at pH 8. Descending order of the stain-effective action on fetal rat cartilage for the basic stains tested was bromophenol blue, aniline blue, Evans blue, methyl violet, trypan blue, and water blue.     

An Automated Technique for Double Staining Rat and Rabbit Fetal Skeletal Specimens to Differentiate Bone and Cartilage

Assessment of chemicals for their potential to cause developmental toxicity must include evaluation of the development of the fetal skeleton. The method described here is an improved and fully automated double staining method using alizarin red S to stain bone and alcian blue to stain cartilage. The method was developed on the enclosed Shandon PathcentreTM, and the quality of specimens reported here will be reproduced only if carried out on a similar processor under the same environmental conditions. The staining, maceration and clearing process takes approximately 6 days. The personnel time, however, is minimal since solutions are changed automatically and the fetuses are not examined or removed from the processor until the procedure is completed. Upon completion of processing, the bone and cartilage assessment of the specimens can be carried out immediately if required. Full evaluation of skeletal development in both the rat and the rabbit is necessary to meet the requirements of safety assessment studies. This method allows this to be accomplished on a large scale with consistently clear specimens and in a realistic time.     

Automated Differential Staining for Cartilage and Bone in Whole Mount Preparations of Vertebrates

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.     

Automated differential staining for cartilage and bone in whole mount preparations of vertebrates

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.     

A Modified Differential Stain for Cartilage and Bone in Whole Mount Preparations of Mammalian Fetuses and Small Vertebrates

Fixation in formol-acetic-alcohol as a prelude to the staining of whole mount vertebrate skeletons with alcian blue and alizarin red S has greatly facilitated the enzyme clearing step of the method outlined by Dingerkus and Uhler. The modified method has been tested on fetal and neonatal mice, and on a variety of vertebrates including bony fish, reptiles, amphibia and birds, and shown to be rapid, reproducible and permanent. The method is not so rapid as that reported by Kimmel and Trammell but is superior at least in certain circumstances. In the present study, optimal results were obtained by fixing in formol-acetic-alcohol for 40 minutes, staining cartilage with alcian blue 8GX, then clearing with trypsin. The time taken to complete the latter step was reduced significantly by incubation at 37 C. The next step was to stain bone using alizarin red S in a weak solution of potassium hydroxide, followed by clearing in a potassium hydroxide-glycerol series.     

A modified differential stain for cartilage and bone in whole mount preparations of mammalian fetuses and small vertebrates

Fixation in formol-acetic-alcohol as a prelude to the staining of whole mount vertebrate skeletons with alcian blue and alizarin red S has greatly facilitated the enzyme clearing step of the method outlined by Dingerkus and Uhler. The modified method has been tested on fetal and neonatal mice, and on a variety of vertebrates including bony fish, reptiles, amphibia and birds, and shown to be rapid, reproducible and permanent. The method is not so rapid as that reported by Kimmel and Trammell but is superior at least in certain circumstances. In the present study, optimal results were obtained by fixing in formol-acetic-alcohol for 40 minutes, staining cartilage with alcian blue 8GX, then clearing with trypsin. The time taken to complete the latter step was reduced significantly by incubation at 37 C. The next step was to stain bone using alizarin red S in a weak solution of potassium hydroxide, followed by clearing in a potassium hydroxide-glycerol series.     

Validation of first annulus formation in red snapper otoliths with the use of an alizarin complexone fluorescent marker

The red snapper (Lutjanus campechanus) is among the most studied reef fish species in the Gulf of Mexico. Several studies have used counts of annuli in sectioned sagittal otoliths to age red snapper. However, interpretation of the putative first annulus has been a major source of debate among otolith readers throughout the Gulf of Mexico. Our objective was to use the chemical marker alizarin complexone to validate the periodicity of first opaque annulus formation in red snapper otolith sections. Juvenile red snapper were immersed in 100 mg alizarin complexone per L seawater solution in November 2005 and then reared in 6000 l circular tanks until July 2006. Otoliths were then removed from the fish and thin sectioned. All experimental otolith sections displayed a distinct fluorescent mark ranging from 0.62 to 0.96 mm from the core when viewed under the microscope with a rhodamine filter. The diffuse opaque annulus was located distally to the alizarin mark in all specimens (ranging from 0.88 to 1.51 mm). The distal position of the presumptive first annulus relative to the alizarin mark in all specimens indicates that this diffuse opaque annulus in red snapper sectioned otoliths forms during the first winter after hatching. Translucent marginal edges of all otolith sections indicate that first opaque annulus formation is completed by mid-July.     

Permanent Preservation of Whole Alizarin Red S Skeletons by Clearing and Embedding in Polyester Resins

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.     

Permanent preservation of whole alizarin red S skeletons by clearing and embedding in polyester resins

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.     

A Simple Method Using Alizarin Red S For the Detection of Calcium in Epoxy Resin Embedded Tissue

This report presents a simple procedure for staining 1-2 μm epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue 0 solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralition. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

A new rapid radiological procedure for routine teratological use in bone ossification assessment: a supplement for staining methods

BACKGROUND: Presently, bone ossification is assessed by the study of single-stained fetal bones (alizarin red-S) or double-stained bones and cartilaginous structures (alcian blue followed by alizarin red-S). Both methods, especially double-staining, are labor-intensive, time-consuming, and provide qualitative information regarding skeleton ossification. Quantitative evaluation of ossification is more difficult and is usually based on determination of calcium and other minerals in the bone by means of atomic absorption spectrometry. Here we introduce a simple new method that allows quantitative determination of skeleton ossification before routine staining examination. METHODS: Fetuses delivered by laparotomy on the 16th and 21st day of gestation as well as 1-day-old rat pups were examined. The fetuses and pups were prenatally subcutaneously exposed to sodium valproate or to physiological saline. Lateral, prone, and supine digital radiograms of each fetus were taken using the Digora-Soredex digital radiography system and the Planmeca Intra intraoral X-ray machine. According to the best visualization, the data concerning vertebra were analyzed. All the fetuses were then routinely double-stained using alcian blue and alizarin red-S. RESULTS: Malformations of axial skeleton (rib, sternum, and thoracic and sacral vertebra) were found in valproate-treated groups. Unlike cartilage malformations, the bone changes were detected in similar frequency in radiological and staining methods. Differences in densities according to the degree of ossification in the vertebral arches and bodies at different levels of the vertebral column, between drug-treated and negative control groups were noted. CONCLUSIONS: The preliminary results suggest that digital radiography examination is a useful method in determining delaying of skeleton ossification not detectable by other methods. It balances qualitative and quantitative aspects of the presently used methods and is also simple, objective, fast, and relatively inexpensive.