Llewellyn's Mordant Blue 3
for Nuclei
This is a substitute for alum hematoxylin in the H&E stain.
Materials
- An aqueous or alcoholic eosin Y solution
- Solution A
Material Variant 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
Material Variant Var A Var B Hydrochloric acid, conc 1 mL 1 mL Ethanol, 70% 99 mL – Distilled water – 99 mL - Solution C
Material Amount Sodium acetate 0.5 g Distilled water 100 mL
Tissue Sample
5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.
Protocol
- Bring sections to water via xylene and ethanol.
- Place into solution A for 5 minutes.
- Rinse well with tap water.
- 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.
- Rinse well with tap water.
- Blue with solution C for about 30 seconds.
- Rinse well with tap water.
- Counterstain with an eosin solution.
- Dehydrate with ethanol, clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – blue
- Cytoplasm – shades of pink
Notes
- 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 substitutes. Mordant blue 3 was one of these, and Llewellyn published three variants of this substitute, both progressive and regressive.
- The 1974 and 1978a formulae are regressive. The 1978b formula is progressive.
- 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.
- Iron alum is ferric ammonium sulphate: FeNH4(SO4)2·12H2O. Use clean crystals.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- 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.