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.
- 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.
- 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.
- To prepare 100 mL of glutaraldehyde/paraformaldehyde:
- 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).
- Heat the parafomaldehyde solution in a fume cupboard to 60°C when the paraformaldehyde dissolves (it is unnecessary to use a thermometer).
- Cool and add 8 mL of EM grade 25% glutaraldehyde.
- Make up to 50 mL with distilled water.
- Make up to 100 mL with 0.2 M phosphate buffer pH 7.4.
- Filter before use in animals.
Fixation of Suspensions, e.g., Viruses, Bacteria, Dissociated Cells
- Centrifuge the suspension at a reasonable speed that will yield a solid pellet of the material under study.
- Add the fixative slowly down the wall of the tube taking care not to dislodge the pellet.
- 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.
- If the pellet resuspends, the pellet can be recentrifuged after each part of the process.
Fixation of Cell Monolayers
- Remove the culture medium, wash in appropriate buffer to remove the excess protein derived from the culture medium and flood with fixative in buffer.
- Wash and osmicate in situ.
- 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 BufferA (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.