Abstract
The lipophilic probe, FM 4-64 does not fluoresce much in water but fluoresces strongly after binding to the outer plasma membrane, providing clear and distinguishable plasma membrane staining. The binding is rapid and reversible. In this protocol vacuoles in yeast cells are stained with the FM4-64 dye, permitting the use of live-cell imaging if needed.
Materials and Reagents
Equipment
Procedure
Notes
This protocol is a pulse-chase procedure designed to label only the vacuole membranes of yeast cells with FM4-64. You may, however, label the membranes of compartments in the entire endocytic pathway (plasma membrane, early and late endosomal membranes, and vacuole membranes) if you continuously pulse the cells with FM4-64 for 60-120 min (i.e., do not chase in label-free medium). Conversely, you may label only the plasma membrane if you add FM4-64 to cells on ice (of course, you will need to maintain the sample at 0 °C during microscopy, which could prove difficult).
Acknowledgments
This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine. Funding to support different projects that have used this protocol has come from NIH – National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, the Pancreatic Cancer Action Network, and the American Heart Association.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Hi Henry, you can find the recipe of the YES medium at https://www.atcc.org/~/media/3907D6168ED64037BCF4693AE75FBA2C.ashxI also copied it below:ATCC medium: 2064 YES MediumYeast extract................5.0 gGlucose.....................30.0 gL-Adenine..................225.0 mgL-Histidine................225.0 mgL-Leucine..................225.0 mgL-Lysine HCl...............225.0 mgUracil.....................225.0 mgDistilled water..............1.0Sterilize medium by 0.2 micrometer filtration.For solid medium:Add 20.0 g agar to 500 ml distilled water and heat to boiling withstirring to dissolve. Autoclave agar solution at 121C for 15 minutes.Cool to 50C. Dissolve nutrient components in 500 ml distilled water andfilter-sterilize. Warm the nutrient solution to 50C. Aseptically combineand mix the agar and nutrient solutions and dispense as required.