An Improved Hematoxylin-Eosin Stain for Sections of Marrow Units

Abstract

The procedure recommended is: Fix “marrow units” (small functional structures of bone marrow) in 10% formol-saline solution for 1–2 hours and dehydrate in 80% alcohol, 95% alcohol and acetone 30 minutes each. Place in fresh 50° and 53°C. paraffin for 30 minutes each. Embed in fresh 53°C. paraffin. Serially section at 5μ thickness and mount with Schleicher's floating solution. Allow to dry for 1 hour in an oven and deparaffinize by passing through xylene I and II, absolute alcohol I and II, and 95% alcohol. Rinse in fresh distilled water and place in dilute Harris' hematoxylin (stock solution 50 ml., distilled water 200 ml.) for 2 to 3 minutes. Rinse well in distilled water and check staining under the microscope. Dip in acid-alcohol 5 times (1 dip to equal about 1 second). Rinse well in weak (0.02%) ammonia water and distilled water. Dip in 2% aqueous phosphotungstic acid about 3 to 5 times (equal to 3–5 seconds). Rinse in fresh distilled water and place in weak ammonia water for 1 minute. Rinse in fresh distilled water I and II. Place in 80% alcohol for 5 minutes and check under the microscope for “blueness” and nuclear differentiation. Place in dilute alcoholic eosin (0.5% alcohol-eosin stock solution 10 parts and 95% alcohol 90 parts) for 1 to 2 minutes. Rinse in 80% alcohol and place for 1 minute in 95% alcohol. Check under the microscope for staining quality. Place in absolute alcohol for 1 minute, alcohol-xylene (equal parts), 10 dips, and xylene I and II. Mount. This hematoxylin-eosin staining schedule brings out minute structural detail of bone marrow tissue heretofore not demonstrable.