Sample type required: Deparaffinized and re-hydrated tissue sections on positively charged slides.
Fixative: 10% NBF or Bouin solution
Alcian blue, pH 1.0
Step | Reagent | Time | Technical Notes |
1 | 0.1N hydrochloric acid (8.2 mL hydrochloric acid concentrate and 991.8 mL distilled water) | Brief rinse | This rinse will protect the subsequent alcian blue solution from pH changes due to the introduction of water. |
2 | 1% alcian blue solution, pH 1.0: 3g alcian blue-8GX dissolved in 300 mL hydrochloric acid, 0.1N | 30 minutes at room temperature or 15 minutes at 37° C |
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3 | 0.1N hydrochloric acid 8.2 mL hydrochloric acid concentrate and 991.8 mL distilled water | Brief rinse | Blot sections dry with fine filter paper after this step. Do not wash with water as this will cause a change in pH and potentially cause nonspecific staining in the tissue section. |
4 | Nuclear fast red solution | Five minutes | Counterstain |
Expected results:
- Sulfated mucins = Pale blue
- Background = Pink to red
- Nuclei = Red
Post staining procedure:
• Tissue sections must be counterstained to fully recognize the presence of alcian blue positive staining. This can be achieved with the use of 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% alcohols, cleared with xylene (or xylene substitute), and cover-slipped.
