*2.10. Histology and Immunohistochemistry*

Cells in monolayer cultures were harvested at the denoted time points for fixation in 4% formalin. Fixed cells were stained with alcian blue for glycosaminoglycans as previously reported [25], with removal of excess stain in double distilled water. The stain was quantitatively estimated using solubilization in 6 M guanidine hydrochloride overnight to measure OD600 nm [25] using a GENios spectrophotometer/fluorometer. Immunohistochemical evaluations were also performed to examine the deposition of type-II, -I, and -X collagen using specific primary antibodies, biotinylated secondary antibodies, and the ABC method with DAB as a chromogen for monitoring under light microscopy (Olympus BX45) [25,44,45]. Control conditions lacking primary antibodies were also evaluated to check for secondary immunoglobulins.
