LipidTOX staining

LipidTOX^TM staining of live larval zebrafish

1. Staining can be performed in six-well dishes or 10 cm tissue culture plates. Alternatively, staining can be done in microcentrifuge tubes or conical tubes. Older larvae ~20 dpf are best stained in 10 cm tissue culture plates. 
2. Calculate the total volume of media/system water that will be required. Use an appropriate volume for the well, dish or tube in which you are performing the staining (ex. 2 ml for 1 well on a 6-well plate).
3. Dilute HCS LipidTOX^TM Green (Thermo Fisher Scientific, H34475) or HCS LipidTOX^TM Red (Thermo Fisher Scientific, H34476) neutral lipid stains in either embryo media or fish system water to 1:5000 and mix thoroughly.
   Note:  For embryos and larvae that have not yet been put onto the nursery, dilute in embryo media; for larvae that have already been transferred to tanks in the fish facility, dilute in system water.
4. Transfer the appropriate volume of diluted LipidTOX^TM staining solution into the wells and dishes as needed. 
5. Transfer the larvae into the diluted LipidTOX^TM staining solution. 
   Note: Smaller larvae can be pipetted with a wide tip glass pipet (Duran Wheaton Kimble, 63A53WT).  Older, larger larvae will need to be removed from system tanks with a fine-mesh net, rinsed in fresh system water and then gently transferred to the staining solution.  The use of a disposable plastic transfer pipet with the tip cut off is helpful here (VWR 414004-004). 
6. Incubate the fish in the diluted LipidTOX^TM staining solution for a minimum of 2 hours prior to imaging, preferably in the dark.
7. Mount and image as desired, both low-magnification whole mount imaging and confocal imaging is possible. No rinsing is necessary.

According to the Thermo Fisher HSC LipidTOX Neutral Lipid Stains manual (https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2Fmp34475.pdf), the approximate fluorescence excitation/emission maxima are:  495/505 nm for LipidTOX^TM Green neutral lipid stain and 577/609 nm for LipidTOX^TM Red neutral lipid stain.