Safranin Red O Stain

Dominik Haudenschild, UC Davis 2009, From ORL Histology Notebook

• Purpose: Stain for Cartilage in decalcified, Paraffin-embedded bone as well as in mineralized, plastic-embedded bone sections.

• Reagents:

1. Fast Green FCF (0.1%).................... 0.50 gram

Distilled Water....................................... 500 ml

2. Glacial Acetic Acid (1%)........................ 10 ml

Distilled Water.................................. 1000 ml

3. Safranin Red (0.1%)........................ 0.50 gram

Distilled Water.................................... 500 ml

• Procedure:

1. Deparaffinize sections in 2 changes of xylene for 5 minutes each. Deplasticize sections in 2 changes of xylene at 55°C for 1-2 hours. Hydrate in 2 minute changes of 100%, 100%, 95%, 70% ethanol in water.

2.Stain in 0.1% Fast Creen FCF for 2 minutes

3.Differentiate slides in 1% acetic acid for 10 dips

4.Stain in 0.1% SafraninRed O for 10 minutes

5.Rinse slides in 95% ethanol for 1-2 minutes

6.Dehydrates slides in 95%, 100%, 100% ethanol for 3 minutes each

7.Clear in 2 changes of Xylene for 3 minutes each

8.Mount with a cover-glass

• Results:

Cartilage - Red

Mineralized Bone - Green

Note: Hematoxylin can be added to the procedure between steps 1 and 2.