TEM Fixation - Protocols - Microscopy

TEM Buffers and Fixation Methods for General Users

Fixation of Tissue

There are two methods of fixation of tissue from organs: cardiovascular perfusion and immersion fixation. Paraformaldehyde is a monoaldehyde and penetrates faster than glutaraldehyde, but results in poorer ultrastructure. A solution is to use a mixture of both aldehydes as in perfusion fixation.

  1. For immersion fixation, use 2.5% glutaraldehyde (must be EM grade) in 0.1M buffer. The time of fixation is dependent upon the dimensions of the sample to be fixed. The largest recommended size is 1 mm3, when there is optimal penetration. Proceed to Step 4.
  2. For perfusion fixation, use 2% glutaraldehyde and 2% paraformaldehyde in 0.1M buffer. The conditions depend upon the animal, its age and the organ required.
  3. To prepare 100 mL of glutaraldehyde/paraformaldehyde:
    1. Add 2 g paraformaldehyde to approx 35 mL distilled water + 0.5 mL of approx. 1 M NaOH (make this each time by dissolving 5 pellets of NaOH in approx 5 mL distilled water).
    2. Heat the parafomaldehyde solution in a fume cupboard to 60°C when the paraformaldehyde dissolves (it is unnecessary to use a thermometer).
    3. Cool and add 8 mL of EM grade 25% glutaraldehyde.
    4. Make up to 50 mL with distilled water.
    5. Make up to 100 mL with 0.2 M phosphate buffer pH 7.4.
    6. Filter before use in animals.
  4. Fixation at room temperature for 1 hour is a good start point. Once the tissue is fixed and dissected, it is washed by aspiration 3 × 5 min in fixation buffer and cut into smaller blocks of 1 mm3. All the remaining procedures are carried out by aspiration. The blocks can be stored in fixation buffer at 4°C for 1–2 days up to one week before subsequent processing. Keeping the samples in 4°C and sending them to Microscopy Core Facility.

Fixation of Suspensions, e.g., Viruses, Bacteria, Dissociated Cells

  1. Centrifuge the suspension at a reasonable speed that will yield a solid pellet of the material under study.
  2. Add the fixative slowly down the wall of the tube taking care not to dislodge the pellet.
  3. Allow to fix for 10 min at RT and then release the pellet using a wooden cocktail stick and leave for a further 20 to 60 min. The material can now be treated as tissue blocks.
  4. If the pellet resuspends, the pellet can be recentrifuged after each part of the process.

Fixation of Cell Monolayers

  1. Remove the culture medium, wash in appropriate buffer to remove the excess protein derived from the culture medium and flood with fixative in buffer.
  2. Wash and osmicate in situ.
  3. Remove cells by scraping from the support using Parafilm-coated spatula or other appropriately shaped implement. Treat as for suspensions.

Preparation of 0.2 M Sorensons Phosphate Buffer

A (mL)..........B (mL)..........pH
Solution A: NaH2PO4.2H2O, mol wt 156.01, 3.12 g in 100 mL H2O.
Solution B: Na2HPO4, mol wt 141.96, 2.84 g in 100 mL H2O.
Note: pH 7.4 buffer fits most of samples.

0.2 M cacodylate buffer.

21.4 g Na cacodylate in 250 mL distilled water. Adjust the pH to 7.4 with approx 8 mL of 1 M HCl and make up to a final volume of 500 mL.