Prepared by
ROY ELLIS
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011
Principle
The dialdehydes formed by treatment with periodic acid from certain carbohydrates, will selectively reduce an alkaline hexamine-silver salt mixture. This method is especially suitable for renal glomerular basement membranes. This method helps determine the thickness of glomerular basement membranes and mesangial matrix.
Method
1. Deparaffinise sections with xylene then take through alcohols to distilled water.
2. Rinse well in distilled water.
3. Treat with 0.5 % periodic acid 5 mins
4. Rinse well in distilled water.
5. Place in freshly filtered silver methenamine solution at 58 - 60 degrees C. Check the slide microscopically after 20 minutes then every 5 minutes until the basement membrane is well demonstrated.
6. Rinse well in distilled water.
7. Tone in 0.2% gold chloride 2 mins
8. Rinse in distilled water.
9. Treat with 2% sodium thiosulphate (hypo) 2 mins
10.Wash in running tap water.
(NOTE:At this point sections can be differentiated using 0.5% sulphuric acid in 0.2% ferric chloride if overstained.)
Stain sections with either H&E or chromotrope if requested.
Haematoxylin & Eosin Counterstain
1. Stain in Lillie Mayers Haematoxylin solution 5 mins
1. Wash in running tap water 1 min
2. Differentiate in acid alcohol (0.6%) 1-2 sec
3. Rinse in running tap water.
4. Blue in Scotts tap water
5. Rinse well in running tap water
6. Stain in Eosin / Phloxine solution 2 mins
7. Dehydrate, clear and mount in DPX.
Chromotrope counterstain
1. Mordant in bouins fluid at 58 - 60 deg 30 mins
2. Wash well in water until sections loose their yellow colour
3. Treat with 1% phosphotungstic acid (make fresh weekly) 1 min
4. Wash well in water.
5. Stain with Chromotrope 2R 15 mins
6. Rinse quickly in distilled water.
7. Dehydrate in alcohols, clear in xylene and mount in DPX
Results
- Basement membranes ...................................Black
- Background ................................................Red
Reagent Formulae
1. Gomori’s silver methenamine solution
3% hexamine(methenamine)…......................46 ml
5% silver nitrate …...................................2.5 ml
5% borax …............................................6 ml
Ensure that all glassware is thoroughly cleaned with glass cleaning solution and distilled water before measuring any solutions. Filter silver methenamine solution before use.
2. Chromotrope solution
Chromotrope 2R (C.I. 16570) .......................2 g
1/10N hydrochloric acid .............................100 ml
3. 1% phosphotungstic acid
Phosphotungstic acid ................................1 g
Distilled water .......................................100 ml
References
1 Gomori, G. 1946, A new histochemical test for glycogen and mucin. American Journal of Clinical Pathology, V16, p177.
2 Jones, D.B. 1957, Nephrotic Glomerulonephritis. American Journal of Pathology, V33, p313.
3 Lillie, R.D. 1977, H.J. Conn's Biological Stains, 9th edition. Williams and Wilkins
4 Culling C.F.A. Handbook of Hitopathological and Histochemical techniques, Third edition, Butterworths