Shoobridge's Polychrome
for Collagen and Other Structures

Solutions

Lillie's alum hematoxylin

Iron alum
Ferric ammonium sulphate	25	g
Distilled water	500	mL
Glycerol	70	mL
This should be pH 2.0

Acid ethanol
Ethanol, absolute	100	mL
Distilled water	100	mL
Hydrochloric acid, concentrated	1	mL

Formal primer
Picric acid, saturated aqueous	400	mL
Formalin, concentrated	100	mL
This should be pH 2.0

Naphthol yellow primer
Naphthol yellow S	1	g
Distilled water	400	mL
Adjust to pH 2.0 with HCl or NaOH

Tungsto-orange
Phosphotungstic acid	0.5	g
Distilled water	200	mL
Orange G	1	g
Dissolve the phosphotungstic acid.
Then add the orange G.
Adjust to pH 2.5 with HCl or NaOH

Tungsto-acid fuchsin
Phosphotungstic acid	0.5	g
Distilled water	200	mL
Acid fuchsin	1	g
Dissolve the phosphotungstic acid.
Then add the acid fuchsin.
Adjust to pH 2.5 with HCl or NaOH

Tungsto-methyl blue
Phosphotungstic acid	0.5	g
Distilled water	200	mL
Methyl blue	1	g
Dissolve the phosphotungstic acid.
Then add the acid fuchsin.
Adjust to pH 2.5 with HCl or NaOH

Tissue sample
5µ paraffin sections of tissues fixed in neutral buffered formalin for 6-24 hours as the primary fixative, followed by secondary fixation in 0.5% aqueous mercuric chloride for 30 minutes at 37°C, or for several hours at room temperature. Treatment with mercuric chloride may be omitted but the staining results will be inferior.

Method

1. Bring sections to water via xylene and ethanol.
2. Remove mercury pigment and wash well with tap water.
3. Place into iron alum for 10 minutes.
4. Rinse well with tap water.
5. Rinse with distilled water.
6. Place into Lillie's hemalum for 10 minutes.
7. Rinse well with tap water.
8. Differentiate with acid ethanol for 15-30 seconds.
9. Blue with running tap water (50°C).
   Avoid alkaline blueing solutions.
10. Rinse with distilled water (50°C).
11. Stain with naphthol yellow primer or picro-formal primer for 30 minutes at 50°C. Prewarm the solution.
12. Wash in running tap water until differentiated (about 5 minutes at pH 6.5-7.0).
    Only erythrocytes, muscle and fibrin should be yellow.
13. Stain in tungsto-orange solution for 5 minutes at room temperature.
14. Rinse with tap water (pH 7.0 or less) for 15-30 seconds.
15. Stain with tungsto-acid fuchsin for 5 minutes at room temperature.
16. Rinse with tap water (pH 7.0 or less) for 15-30 seconds.
17. Stain with tungsto-methyl blue for 5 minutes at room temperature.
18. Rinse with tap water (pH 7.0 or less) for 15-30 seconds.
19. Drain excess water.
20. Dehydrate rapidly with 3 changes of absolute ethanol.
21. Clear with xylene and mount with a resinous medium.

Expected results

• Erythrocytes  –  Yellow
• Striated muscle  –  Yellow, or bright red if the primer is omitted
• Fibrin  –  Varies: unstained, yellow-red, scarlet, magenta, blueish-red as it ages.
• Fibrinoid  –  Varies: yellow-red, scarlet, crimson.
• Albumin  –  Bright orange-red
• Elastin  –  Clear pink-red
• Nucleoli  –  Bright red
• Platelets: Fresh to medium  –  Unstained to brick red
• Platelets: Old  –  Purplish, grey or pale blue
• Secretory granules (e.g. pituitary)  –  Yellow, red or blue
• Thyroid colloid  –  Red or blue (inactive tends to be blue)
• Bile  –  Emerald to olive green
• Primary amyloid  –  Smoky blue
• Secondary amyloid  –  Pale blue
• Most mucins  –  Pale blue
• Reticulin  –  Blue
• Collagen  –  Deep Blue
• Basement membrane  –  Intense deep Blue

Notes

1. It is strongly advised that the original paper be consulted as it contains a great deal of precise information.
2. The naphthol yellow primer was recommended for routine use, as the picro-formal primer gave redder results.
3. The original paper includes a one-step method using the same dyes. Three stock solutions are prepared. Each solution contains 2 g of dye dissolved into 100 mL of 1% phosphotungstic acid. The staining solution is made by combining equal parts of these three solutions. Staining times are not given and should be established by trial and error.

 

Reference
Shoobridge, Michael P. K., (1983),
A new principle in polychrome staining: A system of automated staining, complementary to hematoxylin and eosin, and usable as a research tool.,
Stain Technology, v 58, page 245

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