Formalin Fixation and Paraffin Embedding of Tissues

Preparation of Cultured Cells for Formalin Fixation

1. If cells are grown on culture plates, grow to 80% confluence (50 million cells). Remove the cells by trypsinization, treatment with EDTA, or scraping (to preserve cell membrane proteins) and place into a centrifuge tube.
2. Centrifuge the cells at 1,000-3,000 RPM for 1 minute. Cool the tube on ice then carefully remove and discard the supernatant.
3. Resuspend the cells in 20 ml PBS and repeat step #2.
4. Resuspend the cells in 20 mls 10% Neutral Buffered Formalin (NBF)(VWR, Cat. #VW3239-4), incubate for 15 minutes at room temperature, and repeat step #2.
5. Resuspend the cells in 20 ml PBS and repeat step #2.
6. Resuspend the cells in 20 ml PBS and repeat step #2.
7. Prepare a 3% agar solution at 56-60°C.
8. Carefully release the cell pellet from the bottom of the tube with a pipette and add 0.5 ml of 3% agar solution.
9. Allow the agar solidify around pellet at room temperature, then remove agar pellet and trim off excess agar from the cells.
10. Place the agar pellet into a tissue cassette.
11. Process the cassette as outlined below under 'Tissue Processing with Tissue Tek VIP Processor'.