The problem is, we get quickly bleaching in acute slices of rat brain, when we work with different nucleus (Acc, DRN and RMTg). The signal in the videos are not clear, we got a noisy signal and, of course, bleaching!
The engineers said us, could we the type of filter for ti get imaging. We already proved it and the problem is not solve it.
Does anyone work with acute slices in rat brain? Has any idea what could be happen?
In addition to Matheus' comments: how about using a brighter Ca2+ indicator such as Fluo-8 or Cal520? We have switched to Cal520 for most single-wavelenght Ca2+ imaging, as we beleieve t to be brighter and less readily extruded from cells (we study cardiac myocytes, not slices though). If you use a brighter indicator, it might help to make your recordings less noisy and reduce bleaching (as you can reduce illumination intensity).
What are you using to perfuse the slices and for how long are you incubating it with the dye?
In addition to Matheus' comments: how about using a brighter Ca2+ indicator such as Fluo-8 or Cal520? We have switched to Cal520 for most single-wavelenght Ca2+ imaging, as we beleieve t to be brighter and less readily extruded from cells (we study cardiac myocytes, not slices though). If you use a brighter indicator, it might help to make your recordings less noisy and reduce bleaching (as you can reduce illumination intensity).
We use to study cardiac myocytes, and we see bleaching whenever we over load the cells with dyes. try to decrease the loading time or the dye concentration, too much dye and too much loading time not always gives you better signal-to-noise ratio.
I would recommend trying cal-520 AM (https://www.aatbio.com/products/cal-520-am?unit=21130) as Martin D Bootman suggested. In my experience as well, cal-520 outperforms the fluo series of dyes. According to the manufacturer, the quantum yield() of cal-520 of ~0.75 is approximately 5x greater than the yield for Fluo-8. The probability that an excited fluorophore will bleach tends to decrease with increasing quantum yield. Also, a higher quantum yield reduces the necessary illumination intensity, further reducing the bleaching rate.
AAT Bioquest provides a protocol for loading cultured cells. If you have cultured cells then replicating their experiments is a good place to start. Otherwise they also provide references to cal-520 AM used in slices. If you still can't get adequate signal following those protocols, then you might want to think about whether your fluorescence microscope is well suited to your project.
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