Safranin-O Staining: A Method to Visualize the Structural Integrity and Cellularity of Cartilage

For cartilage staining, begin with a formalin-fixed, paraffin-embedded cartilage section from a euthanized mouse.

First, immerse the sample in the xylene substitute - a non-hazardous clearing agent that removes the paraffin wax and exposes the dehydrated tissue. Next, treat the sample with decreasing ethanol concentrations to rehydrate the tissue for subsequent staining.

Now, stain the sample with Weigert’s iron hematoxylin - an organic stain containing ferrous ions - and incubate briefly. The hematoxylin molecules form complexes with ferrous ions and penetrate the cell. The ferrous ions act as a mordant and facilitate binding of the hematoxylin to the nucleic acids, imparting them a blue color.

Then, treat the sample with the fast green dye - a counterstain that binds to the connective tissue and imparts a green color. Thereafter, treat the sample with an acetic acid solution to stabilize the dye molecules. Lastly, stain the sample with safranin-orange - a cationic dye - and incubate.

The positively charged safranin molecules migrate toward the negatively charged proteoglycans in the cartilage and bind to them, imparting a red color. Subsequently, mount the slide in a resin medium and visualize it under a microscope.

Healthy cartilage appears intensely red due to the proteoglycan-rich extracellular matrix, while unhealthy cartilage appears less red with signs of tissue damage.

To perform Safranin-O staining, first deparaffinize the slides in oxylene for three to five minutes, repeating the oxylene bath twice. Then, hydrate the slides in an ethanol gradient, ending with distilled water. Then, place the slides through Weigert's Iron Hematoxylin solution, a 0.05% Fast Green solution, a 1% acetic acid solution, and a 0.1% Safranin-O solution. Now, mount the slides using a resinous medium.