nile red staining protocol

It has environment-sensitive fluorescence. Nile Red was imaged with 561‐nm excitation and detected at 600–650 nm. Overview ab228553 Nile Red Staining Kit is designed for the quantitative, fluorometric detection and measurement of intracellular lipid droplets using fluorescence microscopy, flow cytometry or a fluorescence microplate reader. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. ab228553 Nile Red Staining Kit 3 1. To combine the Basic Fuchsin or Nile Red staining with Direct Yellow 96: 1. Furthermore, we explore the potential of Nile Red staining in combination with photoluminescence spectroscopy to identify the polymer type and to … Cells detach from the plate, thus losing cells Add 150µl of Nile Red/isopropanol solution. The Nile red staining method used in this study clearly demonstrated the presence of phospholipids. Wrap tubes in foil so that staining occurs in the dark. Move the seedlings to 0.1% Direct Yellow 96 (in ClearSee) and stain for 1 hour; 5. Let worms stain for 30 minutes at room temperature with gentle rocking. The solution was exchanged at least three or four times during washing. Wash for 30 min in ClearSee with gentle agitation; 3. Nile red is intensely fluorescent in a lipid-rich environment while it has minimal fluorescence in aqueous media. Prior to staining the cells are washed with PBS 2-3 times. It should be stored at 4°C in the dark. To make the Nile Red/isopropanol solution, add 6µl of Nile Red stock (0.5mg/ml Nile Red dissolved in acetone) per 1ml of 40% isopropanol. This method proved to be a simple, rapid, and inexpensive procedure that can be used as a sentinel screen for phospholipids in future studies where drug-induced RESOURCES Troubleshooting Problem Possible Causes Recommended Solutions No staining or poor staining Cells are not healthy or dead Use only healthy cells No difference among treatments, including positive control A. For Nile Red staining 0.05% Nile Red in ClearSee solution was prepared and the seedlings were stained overnight. Here, we compare the staining protocols published prior to 2019 and propose an optimized staining protocol. It works for both live/fixed cells. Nile Red is prepared in acetone to make a working concentration of 1000 ug/mL. I find that chemistry is often taught poorly or without a purpose. Clear and stain the seedlings with Basic Fuchsin or Nile Red as described above; 2. Wash once more in ClearSee for at least 1 h; 4. Next, the seedlings were washed for 30 min with gentle shaking. 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