Alcian Blue pH 2.5
Purpose
To demonstrate acidic mucosubstances (hyaluronic acid, sialomucins, and some sulfomucins).
Principle
Alcian blue is a group of polyvalent basic dyes that are water-soluble. The blue color is due to the presence of copper in the molecule. The 3% acetic acid solution (pH 2.5), Alcian blue stains both sulfated and carboxylated acid mucopolysaccharides and sulfated and carboxylated sialomucins (glycoproteins). Alcian blue is believed to form salt linkages with the acid groups of acid mucopolysaccharides.
Solutions
3% Glacial Acetic Acid (prepare in fumehood)
- Concentrated Acetic acid 30 ml
- Distilled water 970 ml
Solution is stable for 6 months.
CAUTION: Contains acid
Wear gloves, goggles and lab coat. Add acid to water. Call Hazmat (911) for spills over 500ml otherwise contain spills with ACID SPILL KITS.
1% Alcian Blue Solution
3% glacial acetic acid 500 ml
Alcian blue-8GX 5 gm
- Mix, adjust pH to 2.5, using acetic acid.
- Filter and add a crystal/grain of thymol
Solution is stable for about 3 months.
CAUTION: Contains acid, avoid contact and inhalation of dye.
Prepare stain in fume hood. Avoid contact and inhalation of dyes.
Nuclear Fast Red (Kernechtrot)
Aluminum sulfate 25 gm
Distilled water 500 ml
Nuclear fast red 0.5 gm
- Dissolve aluminum sulfate in DH2O.
- Add nuclear fast red and dissolve with aid of heat.
- Cool, Filter and add a crystal/grain of thymol (prevents bacterial growth)
Stable for 2 months.
Caution: Irritant avoid contact and inhalation
Safety
Acetic acid: Irritating to respiratory system. Target organ effects on respiratory system by inhalation. Corrosive.
METHOD
- Hydrate slides to distilled water.
- Place slides in 3% acetic acid for 3 minutes.
- Place slides in alcian blue solution for 30 minutes at room temperature or 15 minutes in a 37ºC waterbath.
- Rinse briefly (3-5 dips) in 3% acetic acid (use same coplin jar as step # 2) to remove excess alcian blue.
- Wash in running water for 2 minutes.
- Place slides in nuclear-fast red for 15 minutes
- Wash in tap water for 1 minute (if this step missed, slides will be cloudy)
- Dehydrate quickly in two changes of 95% then absolute, clear, and mount.
Results
Acid mucins/mucosubstances (sulfated and carboxylated): Blue
Nuclei: red
Notes
- Alcian blue pH 2.5 has bench life of approximately 3 months.
- Filter each time before use.
Control
Bowel mucosa
References
IWK. (2007). IWK Laboratory Histology Staining Manual, Special Stains.
Carson, F. L., & Cappellano, C. H. (2015). Histotechnology: A Self-Instructional Text (4th ed.). American Society of Clinical Pathologists Press.
