Paraffin Embedding of Tissue Samples

SOP

• SOP - Carcinogens
• SOP - Flammables and Combustibles
• SOP - Formalin and Paraformaldehyde
• SOP- Irritants

Paraffin embedding protocol

1. Harvest tissue from animal, place into cassette and submerge in 10% neutral buffered formalin for 24-48 hrs (depending on thickness of sample).
2. Move cassettes into 70% ethanol for 24 hrs. Samples can be left in 70% for long term storage, if required. At this step samples can also be taken to the Rogel Cancer Center's Tissue and Molecular Pathology Core for the remaining steps
   1. Note that the subsequent dehydration steps involve many long incubation periods and therefore should be started first thing in the morning (unless you plan on being in the lab overnight!).
3. Turn on wax pots.
4. Move cassettes into 75% ethanol for 30 minutes.
5. Move cassettes into 95% ethanol for 75 minutes. Repeat this step a second time, using fresh 95% ethanol.
6. Move cassettes into 100% ethanol for 60 minutes. Repeat this step twice more, using fresh 100% ethanol each time.
   1. Some protocols suggest that the first 100% ethanol wash can be left over night if time is an issue. However, it must be noted that extending the dehydration process can result in alterations to the morphology of your tissue.
   2. During these incubation steps, prepare beakers with 58-60 deg C paraffin (the melting point of paraffin is 58 C; many embedding procedures recommend that the paraffin to be 2 C above the melting point for best results).
7. Move cassettes into Citrosolv for 30-60 minutes. Repeat this step a second time, using fresh Citrosolv.
8. Move cassettes into 60 deg C paraffin for 60 minutes. Repeat this step twice more, using fresh paraffin each time.
   1. Some protocols suggest that the second paraffin incubation step can be extended over night. However, this may increase the risk of the tissue cracking during sectioning.
   2. During these incubation steps, turn on the paraffin wax machine (Link Building, Room 311).
9. Immediately prior to embedding, spray wax mold with "Mold Grease" and coat the bottom layer of the mold with melted paraffin.
10. Take your sample and orient it on top of the base wax as desired. Partially cover with more paraffin.
11. Place pathology cassette on top of the mold and completely fill the mold.
12. Place mold with sample on "Cold Side" of embedding station .
    1. The paraffin will solidify in 10-15 minutes.
13. Remove embedded sample from the mold (should just slip out).
14. Store at room temperature until ready to section.

Wash Solution Table

Solution	Time
70% ethanol	Overnight/24 hours
75% ethanol	30 min
95% ethanol	75 min
95% ethanol	75 min
100% ethanol	60 min
100% ethanol	60 min
100% ethanol	60 min
Citrosolv	30 min
Citrosolv	30 min
60 degree paraffin	60 min
60 degree paraffin	60 min
60 degree paraffin	60 min or O/N