Koshland Web Site/Methods
Fixation Technique
1. Spin cells in eppendorf and remove supe
2. Add 100 microliters of paraformaldehyde and vortex
3. incubate at RT for 15’
4. Spin cells and remove supe
5. Wash once in KPO4/sorbitol (0.5-1 mls).
6. resuspend in small volume of KPO4/sorbitol
7. Store cells in refrigerator for up to one month.
8. Sonicate cells for 3 seconds on setting 3 before doing microscopy.
9. Pipette one to three microliters of cells onto coverslip (if you put more on, the cells will swim around).

1. Dissolve 4 g of paraformaldehyde in less than 100 mls warm water (you will need to add a drop or two of NaOH to get it to go into solution). NOTE: paraformaldehyde is bad for you, so cover the top of the beaker while you mix this up.
2. Bring the volume to 100 mls with water
3. Add 3.4 g sucrose and dissolve
4. filter sterilize and store at 4 degrees

68 g per 500 ml water (warm the water first)
filter sterilize

87 g per 500 ml water
filter sterilize

2M sorbitol
182 g per 500 ml water (heat water)
filter sterilize

1M Potassium phosphate, pH 7.5
83.4 ml K2HPO4
16.6 ml KH2PO4

60 ml 2M sorbitol
10 ml 1M potassium phosphate
30 ml water
Comments: From Sue Biggins
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