1 Describe the mechanism of Alizarin Red staining of osteobl

1.   Describe the mechanism of Alizarin Red staining of osteoblasts and osteocytes. What does the dye actually adhere to?
2.   Describe the mechanism of Oil Red O staining of adipocytes. What was hematoxylin used for?
3.   Describe the mechanism of Alcian Blue staining of chondroblasts and chondrocytes in the micromasses.

Solution

Alizarin red S (ARS) staining is used to identify the calcium deposits in tissue sections.The dye is very versatile that it can be extracted from the stained monolayer.At first the monolayers in 6 well plates should be washed with PBS and fixed in 10% formaldehyde at room temperature for 15min.The monolayers were then washed twice with distilled water prior to addition of stain with 1ml 2% Alizarin Red S solution for 15 minutes at room temperature.We have to adjust the pH of ARS solution to 4.1-4.3 with 0.5%ammonium hydroxide.Then the plates were incubated at room temperature for 20min with gentle shaking.After aspiration of the unincorporated dye,the wells are washed four times with distilled water.Stained monolayers were observed under microscope.

Calcium forms an Alzarin Red S-calcium complex it precipitates with alizarin, when immersed in alizarin calcium stains in red immediately.Thus both pure calcium and calcium in bones and other tissues can be stained.