HandE

Harris Hematoxylin & Eosin-Xylol
Staining Protocol
(Modified from various sources)

Working Notes: Combine water and acid in a flask and add carmine until no more will go into solution. Bring contents of the flask to ca. 95 C over a boiling water bath, but DO NOT BOIL. Cool and filter. (A double drip coffee filter works well.) Measure stain volume and add an equal volume of 70% EtOH. Store away from excessive heat and light. Keeps for years. Discard stock stain when stained cytoplasm begins to appear muddy or stained specimens appear bluish-purple.

Glacial Acetic Acid
Distilled water
Carmine Alum Lake
70% Ethanol

250 ml
250 ml
5 g
500 ml

Semichon's Acid Carmine

Procedure

Primary Fixation: Fix specimens or tissues and affix them to slides or coverslips. (AFA or Bouin's Fluid are preferable

Reagents

Eosin Staining

Alcohol Option

Xylene Option

Eosin Counterstain: Stain with eosin for 15 sec - 2 min. Agitate gently before allowing specimens to stain in order to produce an even stain.
Dehydration: Dehydrate in a graded ethanol series: 50%, 70%, 95% (3 min each); 100% (3 changes, 3 min each). This step also removes excess eosin - timing in 95% EtOH depends upon the specimen.
Clearing: Clear in a graded xylene or toluene series: 50% xylene/50% Absolute alcohol (3 min); 100% xylene (3 changes, 3 min each).
Mounting: Mount in Damar Balsam (Damar Xylene).

Dehydration: Dehydrate in a graded ethanol series: 50%, 70%, 95% (3 min each); 100% (3 changes, 3 min each).
Clearing and Counterstaining: Clear in a graded xylene or toluene series: 50% xylene/50% Absolute alcohol (3 min); 100% xylene (3 changes, 3 min each). Use Zelmer’s Eosin-Xylol in place of the SECOND change of pure xylene.
Mounting: Mount in Damar Balsam (Damar Xylene).

1. This is an excellent staining procedure that produces satisfactory results regardless of the skill level of the technician as long as destaining is sufficient.
2. In many cases (especially sectioned material), the 50% EtOH rinses can be dropped.
3. For gregarines at least, Zelmer’s Eosin-Xylol provides the best, most predictable stain. Use this method rather than the standard eosin stain before dehydration.

Notes

Working Notes: (1) Dissolve the hematoxylin in the alcohol and (2) the alum in the water with heat. (3) Remove from heat and mix the two solutions. (4) Bring to a boil as rapidly as possible (limiting this heat to less than 1 min and stir often). (5) Remove from heat and add the mercuric oxide slowly. (6) Reheat to a simmer until it becomes dark purple. (7) Remove from heat immediately and plunge the vessel into a basin of cold water until cool. (8) The stain is ready to use as soon as it is cool. Add 2 - 4 ml of glacial acetic acid per 100 ml solution to increase precision as a nuclear stain. Filter before use.

Hematoxylin Crystals
100% EtOH
Ammonium or Potassium Alum
Distilled Water
Mercuric oxide (red)

2.5 g
25 ml
50 g
500 ml
1.25 g

Harris’ Hematoxylin

Working Notes: Dissolve the eosin in the water. Add the alcohol to complete stock solution. Dilute the alcoholic stock 1:3 with 80% EtOH to produce a Working Eosin Solution just before use. Add 0.5 ml glacial acetic acid to each 100 ml of stain and stir before use.

Eosin Y, water soluble
Distilled Water
Alcohol, 95%

1 g
20 ml
80 ml

1% Stock Alcoholic Eosin

Working Notes: none.

Alcohol, 70%
Hydrochloric Acid, concentrated

500 ml
5 ml

1% Acid Alcohol

Working Notes: none.

Tap water
Ammonium hydroxide, 28%

1000 ml
2 - 3 ml

Ammonia Water

Working Notes: none.

Lithium Carbonate
Distilled water

1 g
100 ml

Saturated Lithium Carbonate

Working Notes: Dissolve the eosin in the water. Add the hydrochloric acid and mix well. Allow the precipitate to settle overnight and decant the clear supernatant. Add Xylol to the precipitate and shake gently or stir with a glass rod; avoid an emulsion. During this process the free acid of eosin is dissolved in the xylene layer. Allow to form an organic - inorganic interface and decant the nearly colorless xylol extract. This extract contains the free eosin base and differentially stains organic tissues on contact. This is one of those rare techniques which not only works great but is also a really cool stunt to watch. (Thanks to Derek Zelmer, Emporia State University, for this trick.).

Eosin Y, water soluble
Distilled water
Hydrochloric Acid, concentrated
Xylene

1 g
100 ml
2.5 ml
100 ml

Zelmer’s Eosin-Xylol

fixatives.) Rinse out excess fixative as appropriate (see Fixation Protocols).
Hydration: Hydrate by graded alcohol series 50%, 3 min; Water, 5 - 7 min.
Stain: Stain in Harris Hematoxylin for 15 minutes.
Stain Rinse: Remove excess stain by rinsing in running tap water for 3 min.
Destain and Differentiate: This is the critical step that determines the quality of the final preparation. Destain in 1% acid alcohol until the cytoplasm has only a faint stain but the sharp nuclear stain still remains.
Destain Wash: Briefly rinse in tap water to remove excess acid and halt destain.
Hematoxylin Bluing: Dip in ammonia water or lithium carbonate water until specimens are bright blue (3-5 dips).
Final Rinse: Rinse in running tap water for 10 - 20 min.
Eosin Staining: Eosin counter stain can be added during dehydration (Alcohol Option) or during clearing (Xylene Option). Both work well but they are mutually exclusive techniques. I prefer to stain during clearing using Eosin-Xylol. This technique provides a more predictable stain with superior differentiation.

Dr. R. E. Clopton