McClean: Fixation of Yeast (P. Xu Protocol)
This is the protocol used by P. Xu for fixing yeast cells for GFP fluorescence.
- Yeast cells
- Formaldehyde 37% (Sigma-Aldrich, #252549)
- Phosphate buffered saline??
- 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 at 8000rpm for 1 minute.
- Wash cells with 1X PBS 3 times, spin at 8000rpm for 1 minute each time (can be longer if you need).
- Resuspend cell pellet in 100ul of 1X PBS.
- 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.
Ping Xu 3:12, 25 July 2013 (EDT)
or instead, discuss this protocol.