Rapid Schedules for Koh Clearing and Alizarin Red S Staining of Fetal Rat Bone

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.     

A Modification of Alizarin Red S Technic for Demonstrating Bone Formation: Methods of Mounting, Photographing and Demonstrating the Specimens

This paper shows that by using solutions heated in the incubator during certain stages, the alizarin red S method of staining the ossified centers in embryos has been shortened, with a consequent saving in time.

New methods of mounting the specimens have been evolved and are described in detail.

The technic of photographing mounted and unmounted specimens is outlined and illustrated by diagrams.

Diagrammatic illustrations are provided of the various types of apparatus used, including a plan of the cabinet for demonstrating clearly the smaller embryos mounted between watch glasses. Photographic examples of the results achieved are also shown.     

A Modification of Alizarin Red S Technic for Demonstrating Bone Formation: Methods of Mounting,Photographing and Demonstrating the Specimens

This paper shows that by using solutions heated in the incubator during certain stages, the alizarin red S method of staining the ossified centers in embryos has been shortened, with a consequent saving in time.

New methods of mounting the specimens have been evolved and are described in detail.

The technic of photographing mounted and unmounted specimens is outlined and illustrated by diagrams.

Diagrammatic illustrations are provided of the various types of apparatus used, including a plan of the cabinet for demonstrating clearly the smaller embryos mounted between watch glasses. Photographic examples of the results achieved are also shown.     

Staining of plant Materials Cleared in NaOH

Stains are listed which have proved suitable for staining the epidermis, mesophyll, and sclerenchyma and tracheary elements, respectively, of cleared leaf material of Mouriri and Linociera. Too rapid leaching is avoided by overstaining high in the dehydration series, destaining briefly in the same solvent, and moving through to xylene. Twenty to thirty minutes staining time is generally sufficient. Concentrations and solvents can be varied widely. If destained too much, the material can usually be replaced in the dye with no ill effects. A double stain schedule (Bonnett) of five to ten minutes in 1% Bismarck brown Y in 95% alcohol followed by one to two minutes in 1% fast green FCF in 100% alcohol may be advantageous for thin-walled cells in thick material. It may be preferable to treat thinner material with tannic-acid-iron-chloride followed by safranin (Foster). The effects of bleaches and clearing compounds other than NaOH on staining have not been investigated; however, Dr. Bonnett finds that lactic acid used after NaOH improves clearing and also improves the staining of his combination (above). Mordants can doubtless be used to advantage.     

Alizarin Red S As An Intravital Fluorochrome in Mineralizing Tissues

To avoid interference with weight gain following alizarin red S injections, low doses of an order of 25 mg/kg body weight must be used. Two such doses given 4 days apart to 24-day-old rats produced no visible staining of bone or dentine in undecalcified sections examined by ordinary light microscopy. Under ultraviolet light, however, staining was evident in the form of fine, red fluorescent lines.     

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.     

Decreasing the Time Required for Making an Alizarin Skeleton Preparation

The modification described here involves fixing the material in 10% formalin for at least a week, macerated in 2% sodium hydroxide in an oven at 38°C, stained in dilute alizarin solution and finally stored in glycerin. The whole process for a specimen up to 3 cm. long requires less than a month.     

Recovery and Enhancement of Faded Cleared and Double Stained Specimens

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.     

Recovery and Enhancement of Faded Cleared and Double Stained Specimens

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.     

Automation in Staining of Cytologic Specimens

With continuous automatic staining procedures, it is now possible to stain hundreds of cytologic specimens per hour with a minimum of effort, and with reproducible optimum results. Two automatic and continuous polychrome staining procedures for cervical-vaginal specimens, and two automatic and continuous hematoxylin-eosin staining procedures for abrasion biopsies are presented. The continuous and automatic procedures reduce the number of solutions and the number of procedural steps needed for cytologic staining. They are simpler, less time-consuming, less costly and give fewer chances for error. They assure reproducible staining results even with less experienced technicians. They permit the technician to be occupied with other activities while the greater part of the staining is being done. They require only the part-time use of the Autotechnicon. They permit variation in staining intensity and color differentiation to suit individual preferences. Their use may be of critical importance in the development of effective electronic screening.     

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.     

Double Staining of Skeleton Using Microwave Irradiation

The fetal skeleton double staining method is used to reveal developmental abnormalities in the skeletal system. We used alizarin red S and al-cian blue successfully with microwave irradiation for skeletal double staining. The fixation time was reduced from 4-7 days to 2-2.5 min and the staining time was reduced from 4 days to 23 min.     

Large-scale double-staining of rat fetal skeletons using Alizarin Red S and alcian blue

A critical component in the conduct of a prenatal developmental toxicity study is the evaluation of fetal skeletal development. As the developing rodent fetus is typically evaluated at gestation day 20, at a time when ossification of the skeleton is incomplete, a thorough assessment of skeletal development would include both ossified and cartilaginous structures. Current methods to double-stain the fetal skeleton using Alizarin Red S and Alcian Blue are typically described for small sample sizes or using time allotments for each processing step that are unsuitable for industry. In an industrial setting, there is a need for an effective means to double-stain fetal skeletons on a large scale (i.e., hundreds of fetuses simultaneously). This article describes a method used in our laboratory to stain both fetal bone and cartilage using solutions and procedures on an industrial scale.     

Solution equilibria of Alizarin Red S with Al(III) and Ni(II)

The solution equilibria of Alizarin Red S (NaH₂- ARS, or 9,10-dihydro-3,4-dihydroxy-9,10-dioxo-2- anthracene sulphonic acid monosodium salt) with Al(III) and Ni(II) were investigated. A general procedure for speciation and the determination of equilibria was developed from work by Coleman. and Gampp. The results of the analyses gave a K_H2 for NaH₂ARS of 10^5.71. For the AI(III)/Na₂- HARS equilibrium it was determined that a 1:2 complex was formed in solution with β₂ = 10^12.88. For the Ni(II)/NaH₂ARS equilibrium two species exist, a 1:1 and a 1:2 complex with β₁ = 10^5.35 and β₂ = 10^11.53.     

Staining Problems with Eosin Y: A Note from the Biological Stain Commission

In 1980, eosin Y was the certified dye with which technologists encountered most problems. The specific problem most frequently brought to the attention of the Biological Stain Commission was that solutions of eosin Y formed a precipitate and failed to stain cytoplasm red when used as a counterstain to hematoxylin.     

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.     

The Differentiation of Placoid, Ctenoid and Cycloid Scales by Means of Alizarin Red S

This technic has been used successfully by the author for staining, in situ, the placoid scales of selachians and the smaller forms of cycloid and ctenoid scales of teleosts. Sections of skin are dissected from the ventrum of the specimen, cleaned of fascia and muscle tissue and fixed in a 10% formalin solution. The section is macerated in several changes of a 3% KOH solution until translucent. Staining is accomplished in a fresh 2% KOH solution to which is added a saturated alcoholic solution of alizarin red S. The section remains in the stain for 24 hours. If necessary, the tissue may be quickly destained in acid alcohol (1% H₂SO₄ in 95% alcohol). The skin is dehydrated in cellosolve or the alcohol series and cleared in methyl salicylate. Placoid and teleost scales prepared in this manner are stained a brilliant red. The various parts of the denticle are well differentiated.