Confocal microscopy of whole mount embryonic cartilage: Intracellular localization of F-actin, chick prolyl hydroxylase and type II collagen mRNA

We show that whole mount preparations of embryonic chick sterna can be analyzed with confocal laser scanning microscopy (CLSM). This technique replaces the traditional sectioning of cartilage or culturing of chondrocytes. Whole ‘chunks’ of cartilage can be stained with dyes, used for immunohistochemistry or in situ hybridization. Although other stains have been used, the stains presented include phalloidin and propidium iodide which stain filamentous actin (F-actin) and the DNA and RNA of cells, respectively. Collagen secreting endoplasmic reticulum (ER) was localized with a primary antibody to chick prolyl hydroxylase (CPH) that was detected with a secondary antibody conjugated to FITC. The intracellular localization of type II collagen mRNA was analyzed using in situ hybridization. The cDNA probe specific for the C-propeptide region of the 1 type (II) collagen mRNA was nick translated and labeled with biotin-16-dUTP. Biotin labeled probes were visualized with avidin-FITC. Depending on the intensity of the stain, we were able to analyze approximately 3–10 layers of chondrocytes. Stains penetrated into the cartilage better than antibodies and biotin-avidin labeled cDNA probes. The F-actin was located as bands of filaments in the superficial layers of the cartilage and was associated with the membranes that marked cell boundaries as deep as 10 layers of chondrocytes. The ER stained with anti-chick prolyl hydroxylase was prominent in perinuclear regions of the cells, but the antibody was only able to penetrate 4–5 cell layers. Single label in situ hybridization studies show that chondrocytes are positive for type II collagen mRNA. Similar to the immunohistochemistry, in situ hybridization probes were only able to penetrate 4–5 cell layers. The type II collagen mRNA appeared perinuclear in the chondrocytes, similar to the ER staining pattern.