Melanin Bleach Protocol

 

Prepared byROY ELLIS IMVS Division of PathologyThe Queen Elizabeth HospitalWoodville Road, Woodville, South Australia 5011

Email: roy.ellis@imvs.sa.gov.au

 

 

 

PRINCIPLE

 

Melanin is always intracellular, and varies from pale to dark brown in natural colour.  It is normally found in hair, retina, skin, and substantia nigra of the brain.

 

TECHNICAL POINTS

 

1.      A known melanin containing positive control section must be used.

2.      (step 5) - Check microscopically that the bleach step has been successful on the control.

3.      Steps 6 to 13 may be performed on the staining machine.

 

METHOD

 

1.      Bring sections to distilled water.

2.      Oxidise with potassium permanganate 5 min

3.      Wash in running tap water

4.      Decolourise in oxalic acid     1 min

5.      Wash in running tap water

6.      Counterstain nuclei with haematoxylin 1 min

7.      Wash in running tap water

8.      Differentiate with acid alcohol

9.      Wash in running tap water

10.    Blue in Scotts tap water

11.    Rinse in running tap water

12.    Counterstain with eosin 1 min

13.    Dehydrate, clear and mount.

 

RESULTS

 

                        melanin.....................................colourless

                        nuclei........................................blue

                        erythrocytes................................pink

                        cytoplasm...................................pale pink

 

REAGENT FORMULAE

 

1.      1% aq potassium permanganate

2.      2% aq oxalic acid

 

REFERENCE

 

Lillie, R. 1965.  ‘Histopathologic Technique and Practical Histochemistry’, 3^rd edition, McGraw-Hill