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).


  • 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

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

Guide for clearing time

Clearing step

Clearing agentsTissue sizes
chloroform Ⅰ
chloroform Ⅱ
chloroform Ⅲ
chloroform Ⅳ

※ 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.