CONFOCAL MICROSCOPY OF WHOLE-MOUNT EMBRYONIC CARTILAGE - INTRACELLULAR-LOCALIZATION OF F-ACTIN, CHICK PROLYL HYDROXYLASE AND TYPE-II COLLAGEN MESSENGER-RNA

We show that whole mount preparations of embryonic chick sterna can be analyzed with confocal laser scanning microscopy (CLSM). This techniq ue replaces the traditional sectioning of cartilage or culturing of ch ondrocytes. Whole 'chunks' of cartilage can be stained with dyes, used for immunohistochemistry or in situ hybridization. Although other sta ins have been used, the stains presented include phalloidin and propid ium 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 (CP H) that was detected with a secondary antibody conjugated to FITC. The intracellular localization of type II collagen mRNA was analyzed usin g in situ hybridization. The cDNA probe specific for the C-propeptide region of the alpha1 type (II) collagen mRNA was nick translated and l abeled 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 cD NA probes. The F-actin was located as bands of filaments in the superf icial layers of the cartilage and was associated with the membranes th at marked cell boundaries as deep as 10 layers of chondrocytes. The ER stained with anti-chick prolyl hydroxylase was prominent in perinucle ar regions of the cells, but the antibody was only able to penetrate 4 -5 cell layers. Single label in situ hybridization studies show that c hondrocytes are positive for type II collagen mRNA. Similar to the imm unohistochemistry, in situ hybridization probes were only able to pene trate 4-5 cell layers. The type II collagen mRNA appeared perinuclear in the chondrocytes, similar to the ER staining pattern.