Safranin-O Staining Protocol for Cartilage

Safranin-O staining protocol for cartilage post we have briefly explained about Safranin-O staining method’s principle, requirements, Safranin O staining protocol, result with Label.

Safranin-O Staining Protocol for Cartilage

Safranin O staining protocol, also known as basic red 2, is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. Safranin O staining protocol can also be used for the detection of cartilage, mucin and mast cell granules.

Safranin O staining protocol is used for the detection of cartilage, mucin, and mast cell granules on formalin-fixed, paraffin-embedded tissue sections, and may be used for frozen sections as well. The cartilage and mucin will be stained orange to red, and the nuclei will be stained black. The background is stained green.

Principle

Safranin O staining protocol works by binding to acidic proteoglycans in cartilage tissues with a high affinity forming a reddish orange complex. The binding made cartilage tissues appear red when observed under the microscope.

Requirements

Chemicals

1. Hematoxylin

2. Alcohol

3. Ferric chloride

4. Hydrochloric acid

5. Fast green

6. Glacial acetic acid

7. Safranin O

Equipment’s

1. Light Microscope

2. Coupling jars

Reagents

1. Weigert’s Iron Hematoxylin Solution

Stock Solution A: Hematoxylin: 1 g, 95% Alcohol: 100 ml

Stock Solution B: 29% Ferric chloride in water: 4 ml, Distilled water: 95 ml, Hydrochloric acid, concentrated: 1ml

Weigert’s Iron Hematoxylin Working Solution: Mix equal parts of stock solution A and B. This working solution is stable for about 4 weeks.

2. 0.05% Fast Green (FCF) Solution

Fast green, FCF, C.I. 42053: 0.5 g

Distilled water: 1000 ml

3. 1% Acetic Acid Solution: Acetic acid, glacial: 1 ml in Distilled water: 99 ml.

4. 0.1% Safranin O Solution: Safranin O, C.I. 50240: 0.1 g in Distilled water: 100 ml.

Safranin O staining Protocol

1. Deparaffinize and hydrate slides to distilled water.

2. Stain with Weigert’s iron hematoxylin working solution for 10 minutes.

3. Wash in running tap water for 10 minutes.

4. Stain with fast green (FCF) solution for 5 minutes.

5. Rinse quickly with 1% acetic acid solution for no more than 10 –15 seconds.

6. Stain in 0.1% safranin O solution for 5 minutes.

7. Dehydrate and clear with 95% ethyl alcohol, absolute ethyl alcohol, and xylene, using 2 changes each, 2 minutes each.

8. Mount using resinous medium.

Results

Safranin-O Staining

Nuclei: Black

Cytoplasm: Gray green

Cartilage, mucin, mast cell granules: Orange to red

Further Readings

Reference

  1. https://conductscience.com/safranin-staining/
  2. https://research.mcdb.ucla.edu/Lyons/Protocols_files/SafraninO_staining_for_cartilage.pdf
  3. Safranin’O/’Fast’Green’Stain’for’Cartilage