⑦ Paraffin impregnation

Impregnate the tissue with paraffin to make it hard enough to thinly slice.

The paraffin used for this process

• A mixture of equal amounts of hard paraffin (melting point, 56 − 58 °C) and recycled paraffin (i.e., paraffin scraps generated when making paraffin blocks).

※ Paraffin with a higher melting point is harder. Such paraffin can be thinly sliced; however, blocks of it become brittle. Thus, mixing recycled paraffin with hard paraffin increases block flexibility.

Hard paraffin (melting point, 56 − 58 °C)	Recycled paraffin
Hard and recycled paraffin will always melt in a paraffin wax melter set to 60 °C.
Melting furnace set to 60 °C
Equal quantities of hard and recycled paraffin are poured into a melting furnace set to 60 °C.

Method

Prepare paraffin baths I − IV by melting the paraffin and pouring it into four wide-mouthed containers and placing it into a wax melter set to 60 °C. A lid is not required.
Use filter paper to wipe the tissue slice taken from the chloroform bath and place it in paraffin bath I. Then successively place the tissue into paraffin baths II, III, and IV.
After placing the tissue into paraffin bath III, perform decompression with a vacuum paraffin embedding device for approximately 20 minutes to an hour. Following this, place the tissue into bath IV.
The intermediate solvent in the tissue is removed through the decompression process.

Guide for paraffin impregnation time

• Large (e.g., cerebrum)：8 − 24 hours
• Medium (e.g., cerebellum)：4 hours
• Small (e.g., brain stem, spinal cord)：1 − 3 hours

Paraffin impregnation step

Exchanging the paraffin solution

• The intermediate solvent mixed with the paraffin is volatile and escapes over time. Therefore, it is not necessary to refresh the paraffin solution each time.
• When exchanging the paraffin solution, discard bath I, then sequentially transfer the solution from bath II to I, III to II, and IV to III. Finally, use a new solution for bath IV.
• If the tissue embedded in paraffin contains intermediate solvent, then the intermediate solvent will evaporate when the blocks are thinly sliced, potentially causing the surface to cave in. Ensure that bath IV does not contain any intermediate solvent.
• When rapidly performing paraffin impregnation, if there is a residual odor of intermediate solvent in paraffin bath III, use fresh paraffin in bath IV and sequentially transfer the solution through baths I − III.