Hematox

Heidenhain’s Iron Hemaoxylin
&
Eosin Staining Protocols
(Modified from various sources)

Working Notes: Add Potassium Permanganate Oxidizer dropwise with constant stirring until the staining solution changes from a brown to the color of a deep port wine. (Oxidation is not required when a fully ripen stock is used.) Add the lithium carbonate after oxidation. When the stain begins to produce a muddy cytoplasmic stain, it has become over-ripe and must be replaced (1-2 days). Strength may be altered, but the 0.5% working solution seems to work best.

Alcoholic Hematoxylin Stock
Distilled Water
Potassium Permangante Oxidizer
Lithium carbonate (sat. aq.)

5 ml
95 ml
per vid
3 drops

0.5% Hematoxylin Working Stain

Procedure

Working Notes: To 1 ml of the above solution add ddH20 to make 100 ml. Stable for 24 Hours. Used to rapidly ripen the working hematoxylin staining solution for immediate use when no fully ripened stock solution is available.

Potassium Permanganate
Distilled water

0.2 gm
100 ml

Potassium Permanganate Oxidizer

Working Notes: none.

4% Iron Alum Stock Solution
Distilled Water

50 ml
50 ml

2% Iron Alum Solution

Working Notes: none.

4% Ferric Chloride Stock Solution
Distilled Water

50 ml
50 ml

2% Ferric Chloride Solution

Working Notes: Use violet crystals of iron alum. Yellow crystals have been altered by light. Filter the solution before use. Strengths between 1.5% and 4% may be used, but a 2% solution for mordanting and a stronger (4%) solution for destaining seem to work well.

Ferric ammonium sulfate (iron alum)
Distilled water

4 gm
100 ml

4% Iron Alum Stock

Stock Solutions

Reagents

Working Notes: Will keep for several years if stored in a cool, dark place. The stock is not actually ripe when made, but gives best results after 4-6 months. When no ripe stock is available, the working solution can be artificially ripened by adding 0.02% Potassium permanganate drop-wise to achieve a deep port-wine red color.

Hematoxylin, light crystals
Absolute Ethanol

10 gm
100 ml

Alcoholic Hematoxylin Stock

Working Notes: none.

Saturated aqueous solution.

Lithium Carbonate Solution

Working Notes: Will keep for several years if stored in a cool, dark place.

Ferric Chloride
Distilled water

4 gm
100 ml

4% Ferric Chloride Stock

Primary Fixation: Fix specimens or tissues and affix them to slides or coverslips. (AFA and Bouin's: suitable. Schaudinn's Fluid: preferable.) Rinse out excess fixative as appropriate (see Fixation Protocols).
Hydration: Hydrate by graded alcohol series (50%, 30%, 15%; 3 minutes each) and rinse 3 min in distilled water.
Mordant: Mordant in 2% aqueous iron alum for 45 minutes. Increasing the time to 2-3 hours will sharpen the staining or centromeres and spindle fibers, but no additional resolution is gained after 3 hours. Specimens may be allowed to mordant for up to 12 hours. An iron alum solution may be re-used for mordanting, but never mordant in an iron alum solution that has been used to destain.
Mordant Rinse: Wash out excess mordant be rinsing specimens in distilled water. (Sections, 15-30 seconds; smears, 1 minute) Excess washing will lead to slow and inferior staining.
Stain: Stain in 0.5% Hematoxylin stain solution for 2-3 hours at room temperature. If the entire specimen is black, reduce staining time or samples of variable thickness will destain at very different rates.
Stain Rinse: Remove excess stain by rinsing in distilled water for 5-10 minutes.
Destain and Differentiate: This is the critical step that determines the quality of the final preparation. Destain in 1-2% solution of iron alum or a 1-2% solution of ferric chloride (my preference) until clouds of stain are no longer given off. Continue until the cytoplasm has only a faint stain but the sharp nuclear stain still remains. If specimens become too lightly stained, let them destain completely and return to step 4. End destaining by transfer to distilled water. Aqueous picric acid (saturated) can also be used to destain. It works more slowly and produces a better stain, but it takes time to learn to judge the progress of the destain. There is also little remedy for removing too much stain.
Destain Wash: Rinse the samples for several minutes in tap water. Most tap water is alkaline enough to give the stain a blue-black tint. This color shift can be used to judge the extent of destaining. If additional destain is desired, return to destaining solution. When destaining is satisfactory wash out the destain in distilled water for ca. 30 min (4 water changes, 7 minutes each).
Hematoxylin Bluing: Blue the preparation by washing for 5 minutes in saturated lithium carbonate solution. Some tap water is alkaline enough to blue the stain and make this step redundant.
Dehydration: Dehydrate in a graded ethanol series: 25%, 50%, 70%,95% (3 minutes each); 100% (3 changes, 3-5 minutes each).
Clearing: Clear in a graded xylene or toluene series: 50% xylene/50% Absolute alcohol (5 minutes); 100% xylene (3 changes, 3-5 minutes each).
Mounting: Mount in Damar Balsam (Damar Xylene).
Notes: This is an excellent staining procedure, but it does require a practiced hand. Harris Hematoxylin and Eosin is much more predictable and should be used by inexperienced workers.

Dr. R. E. Clopton