Aim:– To identify collagen and elastic tissue in the same section.

Principle: In the presence of ferric salts (oxidizers) elastic fibers stain with
hematoxylin, along with the nuclei.

Control: skin

Reagents
1. Verhoeff’s solution: Freshly prepared solution gives best result.

Solution A

Hematoxylin                                         5 gm
Absolute alcohol                                100ml

Dissolve hematoxylin with the aid of heat, cool and filter

Solution BFerric chloride                        10gm
Distilled water                                          100ml

Solution C

Iodine                                            2gm
Potassium iodide                        4gm
Distilled water                            100ml

Add 8ml of solution B into 20ml of solution A and then add 8ml of
solution C

2. 2% Ferric chloride solution
3. 1% aqueous solution of acid fuchsin
4. Saturated aqueous solution of picric acid
5. Van Gieson’s stain

Acid Fuchsin 1% (aqueous)                                                    5ml
Saturated aqueous solution of picric acid                           100ml

6. Sodium thiosulphate, 5% (aqueous solution)

Procedure

1. Deparaffinize and take the section to water.
2. Stain in Verhoeff solution until the section is black.
3. Wash in distilled water.
4. Differentiate in 2% Ferric chloride with agitation for few minutes. Check differentiation by rinsing in distilled water. Under      the microscope the elastic fibers and nuclei should stain black and rest of the tissue should be light grey.
5. Put in 5% sodium thiosulphate for 1 minute.
6. Wash in tap water for 5minutes.
7. Counter-stain with Van Gieson’s stain for 1-2 minutes.
8. Differentiate in 95% alcohol.
9. Dehydrate in absolute alcohol two times.
10. Clear in xylene and mount in DPX.

Result:-

Elastic fibres                                  black
Nuclei                                              black
Collagen                                          red 
Other tissues                                  yellow

Note: It is a rapid method but fails to demonstrate fine fibers