3.3.4. Dehydration, clearing, paraffin infiltration and embedding
Complete replacement of water in tissues with paraffin is crucial for obtaining high-quality sections. Firstly, water is replaced with an amphiphilic solvent (e.g. ethanol). Then, this solvent will be replaced with a hydrophobic solvent (e.g. xylene). Finally, the hydrophobic solvent is replaced with paraffin. Longer exposure to each of the different reagents will ensure complete replacement, but may also adversely affect the tissues. Therefore, it is important to optimize conditions, such as the exposure time for each solvent, depending on the type of specimen. Here, we describe conditions for paraffin embedding of the midgut and hindgut of the honey bee.
- Dehydrate the gut tissues by soaking in absolute ethanol for 1 hr at room temperature, repeat twice or more to ensure the replacement of water with ethanol in the tissue.
- Clear dehydrated tissues by soaking in absolute xylene for 20 min at room temperature; repeat twice more for a total of 3 washes.
- Infiltrate paraffin into the tissues by applying liquid paraffin at 65-70˚C, 3 times for 1 hr.
- Put the tissues in melted paraffin in a desired orientation and ‘snap cool’ it in ice-cold water. Do this in a mould (plastic or metal), which has been pre-warmed to the same temperature as the paraffin. Use a Pasteur pipette (also pre-warmed) to pipette melted paraffin into the mould. Add the specimen into the mould filled with paraffin, then ‘snap-cool’ it immediately in the ice-cold water. After leaving it for 5 min in the ice-cold water, the paraffin should have hardened and the block including the embedded tissue can be removed from the mould.
- Determine the direction of your sectioning (cross or sagital) before embedding it in the paraffin block, and then remember the orientation of the sample. Once embedded, the tissue is difficult to see in solid paraffin. Thus, the orientation of the tissue should be marked down for proper orientation of the block when preparing sectioning later.