Alcian Blue pH 1.0 Staining Protocol

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Alcian Blue pH 1.0 Staining Protocol

Sample type required: Deparaffinized and re-hydrated tissue sections on positively charged slides.

Fixative: 10% NBF or Bouin solution

Alcian blue, pH 1.0




Technical Notes


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.


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


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.


Nuclear fast red solution

Five minutes



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.

Alcian blue pH 1.0