university of california, san diego histology shared resources
university of california, san diego histology shared resources
university of california, san diego histology shared resources
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Hematoxylin and Eosin staining <strong>of</strong> Frozen sections:<br />
1. Fix frozen sections in 10% buffered formalin (Fisher SF93-4) for 20 minutes and wash in water<br />
2. stain nuclei by immersing in GILL II HEMATOXYLIN (Surgipath 01522) for 3 minutes.<br />
3. wash in water and immerse in blueing agent (Fisher CS410-4) for 30 seconds, and wash in water<br />
4. Immerse in 95% ethanol briefly<br />
5. Stain cytoplasm by immersing in EOSIN (Surgipath 01602) for 3 minutes and dip once quickly in<br />
water<br />
6. dehydrate by immersing in 1 changes <strong>of</strong> 95% ethanol for 1 min, and then in 3 changes <strong>of</strong> 100%<br />
ethanol (Fisher A962p) for 10 dips each.<br />
7. Immerse in 3 changes <strong>of</strong> Citrosol (Fisher 22-143-975) for 2 minutes each<br />
8. Mount slides with Cytoseal 60 (VWR 48212-154) using glass coverslips (Surgipath 00145)<br />
Hematoxylin / Eosin staining <strong>of</strong> PARAFFIN sections:<br />
1. Deparaffinize by immersing successively, in 2 changes <strong>of</strong> xylene (Fisher X3P) for 10 minutes each<br />
2. Rehydrate by immersing in decreasing concentrations <strong>of</strong> ethanol (Fisher A962p) :<br />
100% for 5 minutes<br />
100% for 5 minutes<br />
95% for 5 minutes<br />
95% for 5 minutes<br />
3. Immerse in water for 5 minutes<br />
4. Follow steps 2 through 8 above.<br />
1. Hematoxylin and eosin stain on frozen sections for Laser Capture microscopy<br />
(American Journal <strong>of</strong> Pathology February 2000 page 446):<br />
1. Thaw frozen sections ( on plain untreated glass slides) one at a time to decrease degradation<br />
2. Fix with 70% ethanol for 10 seconds<br />
3. wash in deionized water<br />
4. immerse in fresh MAYERS hematoxylin for 30 seconds<br />
5. wash in water<br />
6. Immerse in blueing solution for 30 seconds<br />
7. wash in 70% ethanol<br />
8. immerse in eosin for 90 seconds<br />
9. dehydrate sections with two 10 second washes in 95% ethanol and two 10 second washes with<br />
100% ethanol<br />
10. place in xylene for 30 seconds
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