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

Abstract

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.

Effects of fish age versus size on the development of whirling disease in rainbow trout

“…They were euthanized at the time of exposure, fixed in 10% buffered formalin and preserved in 75% ethanol. Cartilage and bone were differentially stained using alcian blue and alizarin red S (Fritzsche & Johnson 1980, Webb & Byrd 1994, Song & Parenti 1995, Ryce 2003. A digital image of each fish ( Fig.…”

Effects of Dietary Phosphate on Ectopic Calcification and Muscle Function in mdx Mice

“…Changes in ectopic calcification in mdx mice skeletal muscle induced by dietary phosphate content were observed using a modified whole body double-staining method involving alizarin red S and alcian blue, which stain bones and cartilage respectively (Dingerkus et al, 1977;McLeod, 1980;Webb et al, 1994). Briefly, 90-day-old mice were sacrificed and fixed in 95% ethanol (EtOH) for 7 days after the skin and organs were removed.…”

Pretreatment with periodate‐oxidized adenosine enhances developmental toxicity of inorganic arsenic in mice

“…Live fetuses were removed from their surrounding membranes, examined for gross abnormalities, and weighed. All fetuses were then stored for 2 or more weeks in 70% EtOH, eviscerated, and double stained with Alizarin red S and Alcian blue by the method of Webb and Byrd (1994). They were then macerated, cleared, and stored by use of the technique of Crary (1962) and examined for skeletal defects.…”