McClean: Fixation of Yeast (Bisaria Protocol)
This is the protocol used by Anjali Bisaria for rapidly fixing yeast cells from chemostat cultures to image for both bud index as well as fluorescence. We found that this protocol preserves both GFP and mCherry fluorescence.
- Yeast cells
- Formaldehyde 37% (Sigma-Aldrich, #252549)
- 0.1M | Potassium Phosphate Buffer pH 7.5
- 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 down 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.
Please feel free to post comments, questions, or improvements to this protocol. Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
*Stephanie S. Steltzer 11:03, 3 August 2015 After troubleshooting we found to preserve the GFP fluorescence best, incubate the tube for just 15min at room temp and after it is spun down, wash once in KPO4 (~0.5-1ml) before resuspending cells in 75-100μL of 0.1M KPO4.Also, cells must be imaged day of.
*Megan N McClean 15:08, 25 July 2013 (EDT) We found empirically that removing the formaldehyde is very important for preserving GFP fluorescence. Don't incubate cells with formaldehyde for more than 20 minutes.
*Megan N McClean I have also used the Koshland Lab protocol (GFP Fixation) and found that it works approximately as well as this protocol.
Anjali Bisaria, Senior Thesis, 2012
Megan N McClean 3:12, 25 July 2013 (EDT)
or instead, discuss this protocol.