⑥ Clearing

Paraffin is immiscible with alcohol. Therefore, the alcohol within the tissues must be replaced with a liquid that is miscible with both alcohol and paraffin, which is termed an intermediate solvent (e.g., chloroform, xylene).

Method

Preparation for the chloroform I − IV baths.
Remove the tissue from the alcohol, wipe it with filter paper or another suitable material, and place it in the chloroform I bath. Place small tissue pieces into a wire mesh basket and then place them into the chloroform bath.
The tissue will float to the surface of the chloroform solution. Therefore, cover the tissue with gauze to prevent it from drying out.
Tissues that are too large to place in the basket can be placed directly into the chloroform bath.
A dish can be used for particularly large pieces of tissue.
Shake the bath by a shaker during this step.

Clearing timeframe

• There is no strict timeframe for chloroform immersion.
• The tissue should be immersed in chloroform for a shorter duration than that for alcohol immersion. Small tissue pieces can be transferred to the next bath in 1 − 2 hours.
• Tissue can also remain in chloroform for a few days to approximately one week without issue. Therefore, this step can be used to adjust the specimen preparation schedule (e.g., adjusting for the embedding schedule or for time periods bookended by consecutive days off).

※ However, leaving tissue in xylene for an extended period of time will cause it to harden.

Guide for clearing time

• Large (e.g., cerebrum)：8 − 24 hours
• Medium (e.g., cerebellum)：4 hours
• Small (e.g., brain stem, spinal cord)：3 hours

Clearing step

※ It is not necessary to replace the chloroform with a fresh solution with each use. Especially the bath I solution can be used 2 − 3 times before disposal. However, the bath IV solution must be replaced with a new solution each time.