⑪ Deparaffinization & hydration
These procedures are performed prior to staining.
Deparaffinization is to remove the paraffin penetrated into the tissue.
After deparaffinization, xylene is removed with 100% ethanol.
Then, slides are hydrated in a series of graded alcohols until water is used.
| １ | xylene I | Deparaffinization |
| ２ | xylene II |
| ３ | xylene III |
| ４ | 100％ ethanol I | Removal of xylene |
| ５ | 100％ ethanol II |
| ６ | 100％ ethanol III |
| ７ | 95％ ethanol | Hydration |
| ８ | 70％ ethanol |
| ９ | Tap water |
Method
- Use a thoroughly clean and dry staining jar or dish for large sections.
- Three changes of xylene is usually sufficent for removal of the paraffin. We prepare xylene I − V solutions because we want to remove the paraffin completely.
- Place the section into baths of xylene I and II for a minimum of 10 minutes and into that of xylene III for a minimum of 5 minutes.
- After the xylene III bath, place the section into ethanol to remove the xylene.
- Gradually reduce the ethanol concentrations from 100% to 95% and then to 70%.
- Place the section into each ethanol bath for 5 − 10 minutes.
- Finally, rinse the section with running tap water to remove the ethanol.
- Replace each liquid by discarding I, replacing I with II, replacing II with III, and renewing III.
Issues to consider with deparaffinization
- When transferring sections between liquids, fully drain the liquid from the section to ensure that the previous bath’s liquid is not transferred to the next bath. It is better to wipe the section with filter paper before transferring it to the next bath. Doing so will minimize liquid spillage and ensure long-lasting liquid. However, ensure that the section does not dry out when draining the liquid.
- Deparaffinization can be quickly completed when the various liquids used are fresh; however, this process takes longer when using liquids that have been reused many times.
- Sections are transparent in xylene; however, they whiten when they are transferred from xylene to ethanol.
- Ethanol can become cloudy when sections are transferred to it from xylene. This means that the ethanol is dirty. If this occurs, then, first, return the section to the xylene III solution and discard the cloudy ethanol as well as xylene I and II. Then, use xylene III as xylene I, and prepare new xylene II and III solutions. Then, repeat the deparaffinization process.
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