Llewellyn's Mordant Blue 3
for Nuclei

Substitute for alum hematoxylin in the H&E stain

Solutions

An aqueous or alcoholic eosin Y solution

Solution A		1974			1978a			1978b
Mordant blue 3		0.25	g		1	g		1	g
Iron alum, 10% aqu.		40	mL		90	mL		5	mL
Sulphuric acid		5	mL		–			–
Hydrochloric acid		–			–			10	mL
Acetic acid		–			50	mL		–
Distilled water		955	mL		860	mL		985	mL

For all variants:–

·  Add the iron alum to the water.

·  Add the mordant blue 3 and mix well.

·  Add the specified acid and filter

The solutions are usable immediately.

They have a shelf life of more than one year

Solution B		var A			var B
Hydrochloric acid, conc		1	mL		1	mL
Ethanol. 70%		99	mL		–
Distilled water		–			99	mL

Solution C
Sodium acetate	0.5	g
Distilled water	100	mL

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Method

1. Bring sections to water via xylene and ethanol.
2. Place into solution A for 5 minutes.
3. Rinse well with tap water.
4. Differentiate with a variant of solution B: –
• For the 1974 formula: var A for 1-3 seconds or var B for 30 seconds.
• For the 1978a formula: var A for about ten seconds.
• For the 1978b formula: do not differentiate. Proceed to step 6.
5. Rinse well with tap water.
6. Blue with solution C for about 30 seconds.
7. Rinse well with tap water.
8. Counterstain with an eosin solution.
9. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  blue
• Cytoplasm  –  shades of pink

Notes

1. At times hematoxylin powder has become difficult, if not impossible, to buy. Due to the importance of this dye in making alum hematoxylin for H&E stains, several other dyes were investigated as substites. Mordant blue 3 was one of these, and Llewellyn published three variants of this substitute, both progressive and regressive.
2. The 1974 and 1978a formulae are regressive. The 1978b formula is progressive.
3. The sections will be blue after solution A. The blueing referred to in step 6 will deepen this, but it's main purpose is to remove any pink staining due to unmordanted dye.
4. Iron alum is ferric ammonium sulphate:  FeNH₄(SO₄)₂·12H₂O. Use clean crystals.

 

Reference
Llewellyn, B. D., 1974
Mordant blue3: A readily available substitute for hematoxylin in the routine hematoxylin and eosin stain.
Stain Technology, v. 49, pp. 347.

Llewellyn, B. D., 1978
Improved nuclear staining with mordant blue 3 as a hematoxylin substitute
Stain Technology, v. 53, pp. 73.