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Formalin Fixation and Paraffin Embedding of Tissues
The following protocols are recommended to fix and embed samples for long term storage and immunostaining. Although various fixatives can be used to preserve tissues, 10% neutral buffered formalin (NBF) has been the most commonly used fixative in pathology. Tissue samples that are fixed in NBF and preserved in paraffin have a nearly indefinite shelf life.
Preparation of Cultured Cells for Formalin Fixation
- 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.
- Centrifuge the cells at 1,000-3,000 RPM for 1 minute. Cool the tube on ice then carefully remove and discard the supernatant.
- Resuspend the cells in 20 ml PBS and repeat step #2.
- 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.
- Resuspend the cells in 20 ml PBS and repeat step #2.
- Resuspend the cells in 20 ml PBS and repeat step #2.
- Prepare a 3% agar solution at 56-60°C.
- Carefully release the cell pellet from the bottom of the tube with a pipette and add 0.5 ml of 3% agar solution.
- Allow the agar solidify around pellet at room temperature, then remove agar pellet and trim off excess agar from the cells.
- Place the agar pellet into a tissue cassette.
- Process the cassette as outlined below under 'Tissue Processing with Tissue Tek VIP Processor'.
Preparation of Fresh or Frozen Tissues for Formalin Fixation
- Trim the tissue so that it is no larger than 4mm x 2.5mm x 2.5mm and place into a tissue cassette.
- Place the tissue cassette into a volume of 10% NBF (VWR, Cat. #VW3239-4) that is at least 10-fold greater than the volume of the tissue, and incubate for 16-24 hours at room temperature.
- Process the cassette as outlined below under 'Tissue Processing with Tissue Tek VIP Processor'.
Tissue Processing with Tissue Tek VIP Processor
Equipment: Tissue Tek VIP 5 Tissue Processor or equivalent, metal sample baskets, plastic displacement blocks.
Materials:
- 10% Neutral Buffered Formalin (VWR, Cat. #VW3239-4)
- 80% Alcohol (Richard Allen, Cat. #8301R)
- 95% Alcohol (Richard Allen, Cat. #8201)
- 100% Alcohol (Richard Allen, Cat. #8101)
- Xylene (Richard Allen, Cat. #6601)
- Paraplast Paraffin pellets (Stat Lab, Cat. #15159464)
- Latex or nitrile gloves
Processor Stations:
| Station #1: | Omit if already formalin fixed |
| Station #2: | Omit if already formalin fixed |
| Station #3 (80% Alcohol): | 45 minutes @ 37°C |
| Station #4 (95% Alcohol): | 45 minutes @ 37°C |
| Station #5 (95% Alcohol): | 45 minutes @ 37°C |
| Station #6 (100% Alcohol): | 45 minutes @ 37°C |
| Station #7 (100% Alcohol): | 45 minutes @ 37°C |
| Station #8 (100% Alcohol): | 45 minutes @ 37°C |
| Station #9 (Xylene): | 1 hour @ 37°C |
| Station #10 (Xylene): | 1 hour @ 37°C |
| Station #11 (Paraffin): | 1 hour @ 60°C |
| Station #12 (Paraffin): | 1 hour @ 60°C |
| Station #13 (Paraffin): | 1 hour @ 60°C |
| Station #14 (Paraffin): | 15 minutes @ 60°C |
For more information about LSBio Protocols contact Technical.Support@LSBio.com
