Alcian Blue pH 2.5 Staining Protocol

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Alcian Blue pH 2.5 Staining Protocol

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

Fixative: 10% NBF or Bouin solution

Alcian blue, pH 2.5




Technical Notes


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.


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% 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.


Running tap water

Ten minutes


Distilled water

Rinse well


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 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 2.5