Llewellyn's Mordant Blue 3
for Nuclei

Substitute for alum hematoxylin in the H&E stain


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:
Dilute the iron alum with the water. Add the mordant blue 3 and dissolve. Add the specified acid, mix and filter The solutions are usable immediately and 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.


  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: –
  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

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  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.


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.



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