Abstract
Staining the endoplasmic reticulum in combination with antibody staining.
Highlights
The ability to stain the entire endoplasmic reticular structure in fixed cells provides greater insight into the interactions between specific proteins of interest and the endoplasmic reticulum. In this protocol we show that a commercial endoplasmic reticulum marker ReZolve-ER can be used to visualise the endoplasmic reticulum following standard immunofluorescence antibody labelling
* CAUTION for best results select a secondary antibody with a fluorophore which is compatible with ReZolve-ER \(ex
To improve ReZolve-ER detection by confocal microscopy use slow collection speeds and high averaging \(2 to 4). Using this protocol the endoplasmic reticulum structure will be visible as a large network like structure throughout the cells, with greatest density in the perinuclear region \(Figure 1A)
Summary
The ability to stain the entire endoplasmic reticular structure in fixed cells provides greater insight into the interactions between specific proteins of interest and the endoplasmic reticulum. This protocol enables researchers to gain insight into protein and endoplasmic reticulum interactions without requiring live-cell imaging capabilities, and enables visualisation of the complete endoplasmic reticulum organelle network in fixed cells. 8. Appropriate secondary antibody \(we recommend Alexa Fluor 647) For adherent cells, seed on sterile coverslips for 24-48 hours. For non-adherent cells, cells can be fixed in suspension and attached to poly-l-lysine coated coverslips before antibody staining.
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