Sample type required: Deparaffinized and re-hydrated tissue sections on positively charged slides.
Fixative: 10% NBF or Bouin solution
Alcian blue, pH 2.5
| Step | Reagent | Time | Technical Notes |
| 1 | 3% acetic acid 15 mL glacial acetic acid and 485 mL distilled water | Three minutes | This rinse will protect the subsequent alcian blue solution from pH changes due to the introduction of water. |
| 2 | 1% alcian blue solution, pH 2.5 5 g alcian blue-8GX dissolved in 500 mL 3% acetic acid | 30 minutes at room temperature or 15 minutes at 37°C |
|
| 3 | 3% acetic acid 15 mL glacial acetic acid and 485 mL distilled water | Brief rinse | Rinse well enough to remove excess alcian blue. This will help to prevent nonspecific staining. |
| 4 | Running tap water | Ten minutes |
|
| 5 | Distilled water | Rinse well |
|
| 6 | Nuclear fast red solution | Five minutes |
|
Expected results:
- Weakly acidic sulfated mucins = Dark blue
- Hyaluronic acid = Dark blue
- Sialomucins = Dark blue
- Background = Pink to red
- Nuclei = Red
Post staining procedure:
- Tissue sections must be counterstained to recognize the presence of alcian blue positive staining fully. This can be achieved using nuclear fast red, which provides a pink-to-red background stain.
- After counterstain, tissue sections should be rinsed well in distilled water, dehydrated with 95% alcohol, followed by 100% alcohol, cleared with xylene (or xylene substitute), and cover-slipped.
