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In vivo microscopy
Protocol: Microscopy and slides
Preparation of slides
- Clean glass slides with 95% ethanol and leave it under the hood for air drying
- Label the slides with the sample name and date</pre>
Methanol Fixing of Cells
- 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.
Some of the samples (seed, LovTAP+dsRed, dsRed only, LovTAP+GFP) were taken to the EPFL BIOP facility for in vivo microscopy imaging. We hoped to detect LovTAP's autofluorescence wavelength (and observe its localization) and red fluorescence for dsRed. We also observed the GFP control.
We used 35 mm plates and filled them with culture samples that had been washed with PBS (and stayed in PBS).
