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.     

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.     

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 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.     

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 silver impregnation technique to demonstrate muscle-bone-cartilage relationships in fishes

A modification of the Winkelmann and Schmitt (1957) technique originally designed to investigate patterns of peripheral nervous innervation is given for demonstration of developing bone growth centers and associated muscle origins and insertions in larval and small fishes. This technique offers a simpler and faster way of obtaining such ontogenetic information than the standard method of double staining with alizarin red S and Alcian blue followed by clearing with potassium hydroxide. Muscles also stain, facilitating the location of their origins and insertions.     

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.     

Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage.

Preparation of small vertebrates cleared after alcian blue staining of cartilage is facilitated by trypsin digestion. Specimens are fixed in formation, washed, skinned, and eviscerated. After staining in a solution of alcian blue in acetic acid-alcohol for 24-48 hours, they are transferred to water through graded alcohols. Excess alcian blue is removed over a period of up to three weeks by changes every 2-3 days of 1% trypsin in approximately one-third-saturated sodium borate. Bony tissues may be stained after this in a solution of alizarin red S in 0.5% KOH. Specimens are bleached if necessary and dehydrated through graded KOH-glycerine mixtures for storage in glycerine. Since alcohol treatment in addition to formalin fixation does not affect results with this method, it should be useful to researchers who want to study the cartilage or cartilaginous skeletons in museum specimens, which are routinely fixed in formalin and stored in alcohol.     

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.     

Clearing and Staining of Embryos for Demonstrating Ossification

The authors have outlined their method for demonstrating fetal ossification whereby the tiniest bones may be studied in their relation to the rest of the skeleton without destroying the specimen. In general the method is that devised by Schultze for clearing the soft tissues with potassium hydroxide. The authors then stain the skeletal system by immersion of the entire specimen in a 0.1% alkalinized aqueous solution of alizarin red S. The dye is removed from the soft parts by decolorization in alcohol-glycerin baths. Details are given for the preparation of the fresh fetus, fixation in alcohol or formalin, clearing, staining, decolorizing, mounting and preservation.     

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.     

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.     

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.     

Double staining technique for rat foetus skeletons in teratological studies.

Cartilage and bone were differentiated using alcian blue and alizarin red S respectively. Anomalies of both cartilaginous and bony parts of the skeleton could be examined.     

Demonstrating the Osseous Skeleton of Human Embryos and Fetuses

Transparent human embryos and fetuses whose osseous skeletons are stained in toto by alizarin red S are successfully prepared when the KOH clearing of the soft tissues and the alizarin staining of the bones are performed simultaneously instead of independently. This modification minimizes the possibility of macerating and staining the soft tissues. Fetuses over 50 mm. CR length are skinned, eviscerated, decerebrated, defatted by dissection, fixed in 95% alcohol, bleached in H₂O₂, cleared and stained simultaneously in an aqueous solution of KOH (from 2% to 10% depending upon the size of the specimen) and .0001 to .00005% alizarin red S (solution has a pale lavender color). This solution is changed periodically to maintain the concentration of the KOH until the clearing of the tissues is complete and of the alizarin until the bones are properly stained. Tissues are dehydrated in increasing concentrations of glycerin and stored in white glycerin plus thymol.     

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.     

Visualization of skeletons and intervertebral disks in live fish larvae by fluorescent calcein staining and disk specific GFP expression

Zebrafish and medaka have become popular models for studying skeletal development because of high fecundity, shorter generation period, and transparency of fish embryo. The first step to study skeletal development is visualizing bone and cartilage. Live animal staining with fluorescent calcein have several advantages over the standard skeletal staining protocol by using alizarin red and alcian blue for bone and cartilage. However, there is no detailed study examining skeletal development of live marine fish larvae by calcein staining. Here we applied calcein staining to examine skeletal development in red sea bream larvae. In addition, green fluorescent protein (GFP) reporter zebrafish was employed to trace lineage analysis of intervertebral disk cells in live fish larvae. Calcein staining of red sea bream larvae successfully visualized development of craniofacial skeletons as well as urinary calculus. Histochemical detection of alkaline phosphatase (ALP) activity revealed that abnormal segmentation of notochord induced by RA during vertebral development in zebrafish. Immunohistochemistry clearly revealed that GFP‐positive cells in intervertebral space was nucleus polposus like cell in twhh‐GFP transgenic zebrafish. It was demonstrated usefulness of calcein and ALP staining and twhh‐GFP transgenic zebrafish for studying skeletal development in live fish larvae.     

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.