Overview
This is the protocol used by Anjali Bisaria for rapidly fixing yeast cells from chemostat cultures to image for both bud index and fluorescence. We found that this protocol preserves both GFP and mCherry fluorescence.
Materials
- Yeast cells
- Formaldehyde 37% (Sigma-Aldrich, #252549)
- 0.1M | Potassium Phosphate Buffer pH 7.5
Protocol
- Prepare eppendorf tubes with 50μL of 37% formaldehyde, one per sample.
- Add 450μL of culture to 50μL of formaldehyde in an Eppendorf tube and mix by inversion. (The goal is to have a final concentration of formaldehyde around 3.7%. So you can adjust the cell and formaldehyde volumes accordingly, as long as you end up with 3.7% formaldehyde).
- Incubate the tube at room temperature for 15-20 minutes.
- Spin gently for 5 minutes at 8000rpm in the small Eppendorf centrifuge.
- Resuspend cells in 75-100μL of 0.1M potassium phosphate
- Store samples at 4°C until you are ready to image.
References
Anjali Bisaria, Senior Thesis, 2012