Paraffin Section Method and Technique

 

 

1. Fix tissues with 10% formalin or other fixatives for 24-48 hours at room temperature. Make sure you have enough fixative to cover tissues.

2. Trim fixed tissues into appropriate size and shape and place in embedding cassettes.

3. Process for paraffin embedding schedule as follow:

   • 70% Ethanol, two changes, 1 hour each

   • 80% Ethanol, two changes, 1 hour each

   • 95% Ethanol, two changes, 1 hour each

   • 100% Ethanol, three changes, 1 hour each

   • Xylene or xylene substitute (i.e. Clear Rite 3), three changes, 1 hour each

   • Paraffin wax (56-58 ºC), two changes, 1 hour and half each

   • Embedding tissues into paraffin blocks.

4. Trim paraffin blocks as necessary and cut at 3-10 um (6 um is commonly used).

5. Place paraffin ribbon in water bath at about 40-45 ºC.

6. Mount sections onto slides.

7. Allow sections to air dry for 30 minutes and then bake in 37-40 ºC oven overnight. NEVER allow baking temperature go higher than 50 ºC.  Otherwise sections may crack, especially 25-50 um thick sections, and result in sections falling off slides during staining.

8. The deparaffinize sections in 2-3 changes of xylene, 10 minutes each. Use three changes of xylene for sections thicker than 25 um.

9. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water.