Team:EPF-Lausanne/Protocol/Microscopy

From 2012.igem.org

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Preparation of slides
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== Preparation of slides ==
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1. Clean glass slides with 95% ethanol and leave it under the hood for air drying
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# Clean glass slides with 95% ethanol and leave it under the hood for air drying
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2. Label the slides with the sample name and date
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# Label the slides with the sample name and date</pre>
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Methanol Fixing of Cells
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== Methanol Fixing of Cells ==
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1. Add 500 µl of PBS in 1.5 ml eppendorf tubes.
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2. Take required volume from cell culture to obtain 1000, 500 and 250 cells and add it to the PBS
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3. Centrifuge at 450 rcf for 5 min
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4. Remove excess PBS manually with a pipet. Make sure you do not touch the cell pellet (may be visible as a tiny dot) (PBS wash 1)
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5. Re-suspend the pellet in 500 µl of PBS.
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6. Centrifuge at 450 rcf for 5 min
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7. Remove excess PBS manually with a pipet. Make sure you do not touch the cell pellet (may be visible as a tiny dot) (PBS wash 2)
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8. Add 20 µl of ice-cold methanol to the cells and re-suspend. Let it rest at -20°C for 10 min
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9. Centrifuge at 450 rcf for 5 min
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10. Remove excess methanol manually with a pipet.
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11. Re-suspend the pellet in 500 µl of PBS.
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12. Centrifuge at 450 rcf for 5 min
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13. Remove excess PBS manually with a pipet.
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14. Re-suspend the pellet in 500 µl of PBS.
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15. Centrifuge at 450 rcf for 5 min
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16. Remove excess PBS manually with a pipet.
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17. Re-suspend the pellet in 10 µl of PBS.
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18. Drop the pellet with PBS on the glass slide
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19. Cover it with cover slip.
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20. Store it at 4°C.
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 +
# Add 500 µl of PBS in 1.5 ml eppendorf tubes.
 +
# Take required volume from cell culture to obtain 1000, 500 and 250 cells and add it to the PBS
 +
# Centrifuge at 450 rcf for 5 min
 +
# Remove excess PBS manually with a pipet. Make sure you do not touch the cell pellet (may be visible as a tiny dot) (PBS wash 1)
 +
# Re-suspend the pellet in 500 µl of PBS.
 +
# Centrifuge at 450 rcf for 5 min
 +
# Remove excess PBS manually with a pipet. Make sure you do not touch the cell pellet (may be visible as a tiny dot) (PBS wash 2)
 +
# Add 20 µl of ice-cold methanol to the cells and re-suspend. Let it rest at -20°C for 10 min
 +
# Centrifuge at 450 rcf for 5 min
 +
# Remove excess methanol manually with a pipet.
 +
# Re-suspend the pellet in 500 µl of PBS.
 +
# Centrifuge at 450 rcf for 5 min
 +
# Remove excess PBS manually with a pipet.
 +
# Re-suspend the pellet in 500 µl of PBS.
 +
# Centrifuge at 450 rcf for 5 min
 +
# Remove excess PBS manually with a pipet.
 +
# Re-suspend the pellet in 10 µl of PBS.
 +
# Drop the pellet with PBS on the glass slide
 +
# Cover it with cover slip.
 +
# Store it at 4°C.
 +
Now the slides can be taken to the BIOP facility at EPFL and imaged with a confocal microscope.
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Latest revision as of 18:18, 26 September 2012

Protocol: Microscopy and slides


Preparation of slides

  1. Clean glass slides with 95% ethanol and leave it under the hood for air drying
  2. Label the slides with the sample name and date</pre>

Methanol Fixing of Cells

  1. Add 500 µl of PBS in 1.5 ml eppendorf tubes.
  2. Take required volume from cell culture to obtain 1000, 500 and 250 cells and add it to the PBS
  3. Centrifuge at 450 rcf for 5 min
  4. Remove excess PBS manually with a pipet. Make sure you do not touch the cell pellet (may be visible as a tiny dot) (PBS wash 1)
  5. Re-suspend the pellet in 500 µl of PBS.
  6. Centrifuge at 450 rcf for 5 min
  7. Remove excess PBS manually with a pipet. Make sure you do not touch the cell pellet (may be visible as a tiny dot) (PBS wash 2)
  8. Add 20 µl of ice-cold methanol to the cells and re-suspend. Let it rest at -20°C for 10 min
  9. Centrifuge at 450 rcf for 5 min
  10. Remove excess methanol manually with a pipet.
  11. Re-suspend the pellet in 500 µl of PBS.
  12. Centrifuge at 450 rcf for 5 min
  13. Remove excess PBS manually with a pipet.
  14. Re-suspend the pellet in 500 µl of PBS.
  15. Centrifuge at 450 rcf for 5 min
  16. Remove excess PBS manually with a pipet.
  17. Re-suspend the pellet in 10 µl of PBS.
  18. Drop the pellet with PBS on the glass slide
  19. Cover it with cover slip.
  20. Store it at 4°C.

Now the slides can be taken to the BIOP facility at EPFL and imaged with a confocal microscope.

Wiki Links

Important pages

Pages

Retrieved from "http://2012.igem.org/Team:EPF-Lausanne/Protocol/Microscopy"