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 Novel Gain-Of-Function Mutation of the Proneural IRX1 and IRX2 Genes Disrupts Axis Elongation in the Araucana Rumpless Chicken

“…Bone and cartilage staining was carried out on E18 Araucana Rp and tailed controls using Alcian blue (Polysciences) and Alizarin red S (Acros Organics) according to standard procedures [33]. Briefly, Embryos were fixed 3×24 hours in 95% EtOH, 100% EtOH, 2×24 h in 100% Acetone.…”

Highly reproducible skeletal deformities induced by administration of β-aminopropionitrile to developing chick embryos

“…Bone and cartilage of embryos were stained in a wholemount preparation according to the protocol reported by Kelly and Bryden (14), with slight modifications reported by Yamazaki et al (15). Briefly, embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.35; PBS) for 2 h at 4°C.…”

“…In the present study, we utilized chick embryos developing in ovo and whole-mount differential staining of cartilage and bone (14,15), allowing direct threedimensional observation of skeletal elements in embryos without the need for dissection. After surveying the skeletal deformities induced by BAPN in embryos, we focused on the reproducibility and exact time course of tibiotarsal deformity, and then examined the alterations present in the bony skeleton of the tibiotarsus using scanning electron microscopy (SEM).…”

Demonstration of Osseous Tissue in Quail Embryo by Alizarin Red Technique

“…Whole mount staining with alizarin red for osseous tissue has been widely used for visualizing the skeletal patterns of embryos of chicks and small adult vertebrates (Yamazaki et al, 2011). The correct evaluation of mineralization is fundamental for the study of skeletal development.…”