Labeling and Visualization of Endoplasmic reticulum using ER Tracker

 ER-Tracker is a cell-permeant, a live-cell stain that is highly selective for the endoplasmic reticulum (ER) that when stained using the protocol provided, the staining pattern is partially retained after fixation with formaldehyde. 


ER-Tracker™ Red (BODIPY™ TR Glibenclamide)
This stain consists of the red-fluorescent BODIPY® TR dye and glibenclamide. Glibenclamide (glyburide) binds to the sulphonylurea receptors of ATP-sensitive K+ channels which are prominent on ER; the pharmacological activity of glibenclamide could potentially affect ER function. Variable expression of sulphonylurea receptors in some specialized cell types may result in non-ER labeling. Excitation: 587nm Emmision 615 nm

ER-Tracker™ Green (BODIPY™ FL Glibenclamide)
This stain consists of the green-fluorescent BODIPY® FL dye and glibenclamide. Glibenclamide (glyburide) binds to the sulphonylurea receptors of ATP-sensitive K+ channels which are prominent on ER; the pharmacological activity of glibenclamide could potentially affect ER function. Variable expression of sulphonylurea receptors in some specialized cell types may result in non-ER labeling. Excitation: 504 nm, Emission: 511 nm

ER-Tracker™ Green (BODIPY® FL Glibenclamide)
ER-Tracker™ Green (BODIPY® FL Glibenclamide) is recommended for live-cell imaging only; fixation with aldehydes or alcohols will inhibit staining. Excitation: 504 nm, Emission: 511 nm, Molecular Weight: 783.10 g/mol

Protocol for ER Tracker Labeling
1.1 Prepare staining solution.
Dilute the 1 mM stock solution to the final working concentration. Use the concentrations of 100 nM–1 μM for ER-Tracker™ Blue-White DPX and ~1 μM for ER-Tracker™ Green and ER-Tracker™ Red dyes. To minimize potential labeling artifacts, use the lowest dye concentrations possible. Best results are obtained when staining is performed in Hankʼs Balanced Salt Solution with calcium and magnesium (HBSS/Ca/Mg, Gibco cat. #14025-092) at 37˚C/5% CO2 . 

1.2 Stain the cells. 
For adherent cells, remove the medium from the culture dish, rinse with HBSS, and add prewarmed staining solution. Incubate the cells for approximately 15–30 minutes at 37˚C. Replace the staining solution with fresh probe-free medium and view the cells using a fluorescence microscope

Fixation and Permeabilization for ER-Tracker™ Blue-White DPX 
2.1 Fix and permeabilize cells. ER-Tracker™ Blue-White DPX signal is only partially retained after formaldehyde fixation. Fix stained cells with 4% formaldehyde for 10–20 minutes at 37˚C. If additional staining will be performed, cells can be permeabilized with 0.2% Triton® X-100 for 10 minutes. 

2.2 Wash and view cells. 
After cells are fixed, perform two 5-minute washes in any suitable buffer and view. 
Fixation for ER-Tracker™ Green and ER-Tracker™ Red Dyes 
3.1 Fix cells.
 If stained cells are to be fixed, fixation is recommended using 4% formaldehyde for 2 minutes at 37˚C. 

3.2 Wash and view cells. 
After fixation, perform two 5-minute washes in any suitable buffer prior to mounting, viewing, or further staining. Permeabilization is not recommended; signal is not retained after permeabilization with Triton® X-100.

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