To dehydrate slides, you typically immerse them in a series of increasingly concentrated alcohol solutions to gradually remove water, preparing them for subsequent steps like clearing and mounting.
Understanding Slide Dehydration
Dehydration is a critical step in many microscopy protocols, particularly when preparing tissue sections or other samples for permanent mounting with non-aqueous mounting media. The process involves replacing the water within the sample and on the slide surface with an organic solvent, usually ethanol, which is miscible with the clearing agents and mounting media used later. This gradual replacement prevents cellular distortion and ensures proper clearing and adhesion of the coverslip.
The Dehydration Protocol
Based on common laboratory practices, a structured approach involving a graded series of ethanol solutions is employed for effective slide dehydration. This method ensures gentle water removal, minimizing damage to the sample.
Here is a step-by-step protocol for dehydrating slides, often used for mounted samples:
- DI Water Immersion:
- Immerse the slides (preferably in a slide boat with a metal handle) in DI (Deionized) water for 30 seconds. This step helps to rinse off any residual aqueous solutions from previous processing steps.
- 50% Ethanol Immersion:
- Transfer and immerse the slides in 50% ethanol for 5 minutes. This initiates the dehydration process by beginning to replace water with alcohol.
- 75% Ethanol Immersion:
- Next, immerse the slides in 75% ethanol for 5 minutes. The increased concentration continues the gradual removal of water.
- 95% Ethanol Immersion:
- Proceed to immerse the slides in 95% ethanol for 5 minutes. At this stage, most of the water has been replaced, and the sample is largely dehydrated.
- First 100% Ethanol Immersion:
- Immerse the slides in 100% ethanol for 5 minutes. This ensures complete dehydration, removing any remaining water.
- Second 100% Ethanol Immersion:
- Finally, immerse the slides in a fresh bath of 100% ethanol for another 5 minutes. A second immersion in absolute ethanol is crucial to ensure thorough water removal and prevent any carry-over of trace amounts of water from the previous bath.
This sequential process ensures that water is removed progressively, minimizing osmotic shock to the cells and preserving the morphological integrity of the specimen.
For more information, you can refer to protocols like the one found at labs.la.utexas.edu/dominguez/2012/01/12/protocol-dehydrating-mounted-slides/.
