Dear all,
I'm doing cell culture with lentiviruses including eGFP.
While this is a very useful way to check transduction efficiency, it does take up a channel with subsequent immunofluorescence microscopy.
I'm considering photobleaching the fixed cells prior to IF staining, does anyone have experience with this?
Sam
Dear Sam,
Frankly speaking this photobleaching approach does not look like a cakewalk. I can imagine how to photobleach specific field of view but this becomes much trickier if we speak about the entire specimen area. To permanently photobleach EGFP within the field of view, one would typically require continuous or time-lapsed irradiation by 488 nm light with power density of at least 1 W/cm2 (higher-better) applied for 1-5 minutes (depending on a microscope setup used). However, after bleaching and IF staining you would need to find precisely the same area of a dish/glass.
The most straightforward way to solve your problem is to use some red- or blue-emitting dye for immunostaining. Alternatively, you can try to chemically bleach EGFP during fixation. Hydrogen peroxide (Article Oxyradical-induced GFP damage and loss of fluorescence
) or zinc salts can be useful. Importantly, chemical bleaching may affect the intracellular structures of interest.Best,
Alex
Agreed with Alexey, photobleaching the cells just seems tricky, if you must use these cells, can you perform imaging in other channels for your immunostains, far red...again assuming you cannot remove the eGFP gene
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