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Eosin in Histology


Most frequently this is an anionic stain, and is most commonly used as the counterstain.

When used properly three shades of pink can be obtained with eosin alone. Erythrocytes, collagen and cytoplasm, muscle or epithelial cells should stain different shades or intensities of pink. Erythrocytes and eosinophilic granules are bright pink to red, cytoplasm and other tissue elements are various shades of pink.

Pale staining with eosin usually results from the PH being over 5.0, it should be from 4.5-5.0. It also may result if the sections are very thin, if they are dehydrated to long, or if they are allowed to stay in the lower concentration alcohols because the water is what decolorizes eosin (that is the reason the step after eosin in any H&E protocol is always 95% alcohol).

If the cytoplasm is over stained it may be because the sections were stained for too long, the eosin may also be too concentrated. The sections may be too thick, or the tissue may have been dehydrated to quickly.

If the eosin staining is not well differentiated (you are not able to see three shades of pink) the slides may need to be rehydrated and then dehydrated again, to expose the eosin to water a second time.

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Oil Red O Stain Protocol

This is one of the stains done on frozen tissues for lipids or fat globules.

Safety equipment: Work under a hood with lab coat, gloves, and glasses.
Use on frozen sections only

1. Hydrate slides in running water———————– 10 minutes
2. Stain in working Oil Red O ————————— 10 minutes
3. Rinse in running water ——————————- 2 minutes
4. Rinse in 60% isopropanol —————————– 5 minutes
5. Rinse in running water ——————————- 2 minutes
6. Counterstain with Harris Hematoxylin —————– 20 seconds
7. Blue in running water ——————————– 5 minutes
8. Coverslip with glycerin jelly or aqueous mounting media

Fat Globules – Orange Red
Nuclei – Blue

• For tissues that do not stay on slides, do not rinse slides in water prior to staining.
• Dextrin must be made for new working solution.
• Dextrin is only good for 1 month.


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