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Solution
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Components
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Concentration
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Comments
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| Phosphate buffered saline (PBS): | NaCl | 8 g/L | Prepare in 1L of sterile-filtered water; adjust pH to 7.2-7.5 |
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KCl | 0.2 g/L |
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KH2PO4 | 0.2 g/L |
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Na2HPO4·2H2O | 1.44 g/L |
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| 4% Formaldehyde/PBS buffer: | A) PBS buffer: | 25 mL/L | Prepare at least once per week; adjust pH to 7.2-7.5 |
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37% formaldehyde solution | 2.7 mL |
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B) PBS buffer: | 25 mL | Heat to 60°C |
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Paraformaldehyde | 1 g/L | White powder (toxic) |
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NaOH | A few microliters | To dissolve paraformaldehyde |
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Fixation procedure
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Comments
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| Add 1.5 mL 4% formaldehyde/PBS buffer to 0.5-cm sample or cell suspension in a 2-mL screwtop microfuge tube. | Samples are taken in 5-mL syringes from cold room on a regular basis and are fixed immediately. | ||
| Keep cold on ice for 1-24 hr. | This can also be done at 4°C or room temperature. | ||
| Centrifuge at 13,000 rpm in the table-top centrifuge. | Optimally at 4°C. | ||
| Remove supernatant, add 1.5 mL PBS buffer, and suspend cells/sediment on whirl mixer. |
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| Repeat these two washing steps to remove formaldehyde. |
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| Centrifuge at 13,000 rpm in the table-top centrifuge. | Optimally at 4°C. | ||
| Remove supernatant and add 1.5 mL of 1:1 mix of PBS buffer/100% ethanol. |
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| Resuspend sample on whirl mixer. | Use samples directly for FISH or keep at -20°C for a maximum of several months. The mixture should remain liquid and therefore must not be frozen at -80°C. Storage at 4°C is also possible for some time. | ||
Note: FISH = fluorescence in situ hybridization.