2.7 HEMATOXYLINS
  1. Gomori's Chrome Alum Hematoxylin
  2. Harris' Hematoxylin and Eosin
  3. Weigert's Iron Hematoxylin and Eosin

Staining Procedures For Plastic Embedded Tissue
Verified at the Applications Laboratory of the Biomedical Division, Sorvall Microtomes

SOLUTIONS:

Bouins's Fluid
75.0ml Picric acid, saturated (approx. 1.22%)
25.0ml Formalin, concentrated
5.0ml Galcial acetic acid
Potassium Permanganate Solution
0.3g Potassium permanganate
100.0ml Distilled water
0.3ml Sulfuric acid
5% Sodium Bisulfite Solution
5.0g Sodium bisulfite
100.0ml Distilled water
Chromium Hematoxylin Solution
50.0ml Hematoxylin, 1%
50.0ml Chromium potassium sulfate, 3%

To 100ml of chromium hematoxylin solution add 0.1g of potassium iodate. Boil until a deep blue. The mixture is ripe immediately and can be used as long as a film with a metallic luster forms on its surface in a Coplin jar. Filter before use.
1% Acid Alcohol
1.0ml Hydrochloric acid
100.0ml Ethyl alcohol, 95%
0.5% Phloxine B Solution
0.5g Phloxine B
100.0ml Distilled water
5% Phosphotungstic Acid
5.0g Phosphotungstic acid
100.0ml Distilled water

STAINING PROCEDURE:

  1. Treat in Bouin's fluid for 12-24 hours.
  2. Rinse in running tap water for 15 minutes.
  3. Treat in potassium permanganate for 1 minute.
  4. Decolorize in sodium bisulfate.
  5. Rinse in running tap water for 10 minutes.
  6. Stain in chromium hematoxylin at 60ºC for 1-2 hours.
  7. Rinse in acid alcohol for 1 minute.
  8. Rinse in running tap water for 5 minutes.
  9. Stain in phloxine B at 60ºC for 2-3 hours.
  10. Rinse in distilled water.
  11. Treat in phosphotungstic acid for 1 minute.
  12. Rinse in running tap water for 5 minutes.
  13. Differentiate in 95% ethyl alcohol for approximately 1 minute.
  14. If section is too red, rinse in 80% alcohol for 15-20 seconds.

Blow dry.
Mount.

RESULTS:

Differential pancreatic islet cell stain. Alpha cells -- pink; Beta cells -- blue; D cells from pink to red are indistinguishable from alpha cells. Cells are pale but useful. Great stain for goblet cells, paneth cells and microvilli.

b. Harris' Hematoxylin and Eosin (Ref 5)

SOLUTIONS:

Harris' Hematoxylin
2.5g Hematoxylin crystals
25.0ml Ethyl alcohol, 100%
50.0g Ammonium or potassium alum
500.0ml Distilled water
1.25g Mercuric oxide (red)

Dissolve the hematoxylin in the alcohol, and the alum in the water by the aid of heat. Remove each from heat and mix the two solutions. Bring to a boil as rapidly as possible. (Limit this heat to less than 1 minute and stir often.) Remove from heat and add the mercuric oxide slowly. Reheat to a simmer until it becomes dark purple; then remove from heat immediately and plunge the vessel into a basin of cold water until cool. The stain is ready for use as soon as it cools. Addition of 2-4ml of glacial acetic acid per 100ml of solution increases the precision of the nuclear stain. Filter before use.
Acid alcohol
1000.0ml Ethyl alcohol, 70%
10.0ml Hydrochloric acid, concentrated
Ammonia Water
1000.0ml Tap water
2-3ml Ammonium hydroxide, concentrated
Eosin-Phloxine Solution
Stock Eosin
1.0g Eosin Y, water soluble
100.0ml Distilled water
Stock Phloxine
1.0g Phloxine B
100.0ml Distilled water
Working Solution
100.0ml Stock eosin
10.0ml Stock phloxine
780.0ml Ethyl alcohol, 95%
4.0ml Glacial acetic acid

Make up working solution as needed. Working solution should be changed at least once a week.

STAINING PROCEDURE:

  1. If sections are Zenker-fixed, remove the mercuric chloride crystals with iodine and clear with sodium thiosulphate (hypo).
  2. Stain in Harris' hematoxylin for 10 minutes.
  3. Rinse in tap water.
  4. Differentiate in acid alcohol for 2-3 quick dips.
  5. Wash in tap water.
  6. Dip in ammonia water until sections are bright blue; 2-3 dips.
  7. Wash in running tap water for 10-20 minutes.
  8. Stain with eosin-phloxine for 2 to 5 minutes at room temperature or 1minute at 60ºC. For even staining results, dip slides several times before allowing them to set in the eosin for the desired time.
  9. Dip in 95% alcohol until excess eosin is removed; 3-5 dips.

Blow dry.
Mount.

RESULTS:

Nuclei -- blue with some methachromasia; cytoplasm -- various shades of pink identifying different tissue components.

c. Weigert's Iron Hematoxylin and Eosin (Ref 2)

SOLUTIONS:

Weigert's Iron Hematoxylin
Solution A
1.0g Hematoxylin (C.C.)
100.0ml Ethyl alcohol, 95%
Solution B
4.0ml Ferric chloride, 29%
95.0ml Distilled water
1.0ml Hydrochloric acid, concentrated
Working Solution
Mix A and B in equal parts. The mixture turns a rich deep blue-violet and is best prepared fresh each time, but it will keep and can be used for several days, sometimes as long as a month.
Acid Alcohol
1000.0ml Ethyl alcohol, 70%
10.0ml Hydrochloric acid, concentrated
Ammonia Water
1000.0ml Tap water
2-3ml Ammonium hydroxide, concentrated
Eosin-Phloxine Solution
Stock Eosin
1.0g Eosin Y, water soluble
100.0ml Distilled water
Stock Phloxine
1.0g Phloxine B
100.0ml Distilled water
Working Solution
100.0ml Stock eosin
10.0ml Stock phloxine
780.0ml Ethyl alcohol, 95%
4.0ml Glacial acetic acid

Make up working solution as needed. Working solution should be changed at least once a week.

STAINING PROCEDURE:

  1. If sections are Zenker-fixed, remove the mercuric chloride crystals with iodine and clear with sodium thiosulphate (hypo).
  2. Stain in Weigert's hematoxylin for 10 minutes.
  3. Rinse in tap water.
  4. Differentiate in acid alcohol to remove background stain for 2-3 dips.
  5. Wash in tap water.
  6. Dip in ammonia water until sections are bright blue; 2-3 dips.
  7. Wash in running tap water for 10-20 minutes.
  8. Stain with eosin-phloxine for 2 to 5 minutes at room temperature or for 1 minute at 60ºC. For even staining results, dip slides several times before allowing them to set in the eosin for the desired time.
  9. Dip in 95% alcohol until excess eosin is removed; 3-5 dips.

Blow dry.
Mount.

Note: Steps 4 to 7 produce a cleaner preparation but may be omitted.

RESULTS:

A good nuclear stain

Warning: Some of the chemicals used for the staining procedures given in this section may be hazardous if misused. For this reason, read and observe all warnings and cautions provided by the manufacturer for each chemical before proceeding with a staining procedure.

Note: In order to prevent sections from loosening from the slides during staining, all sections should be heat-fixed (60ºC to 100ºC) to the slides for a minimum of 2-5 minutes prior to staining, preferably at the time the sections are mounted on the slides.


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