Hi, I'm new to calcium imaging but have been doing electrophysiology (whole-cell patch clamping for a few years). As our imaging system does not have a perfusion system and no carbogen gas available, we are not able to perfuse the brain slice with oxygenated ACSF during imaging. This of course will impact on cell survival due to changes in pH and low oxygenation. Otherwise brain slices were maintained in oxygenated ACSF prior to imaging.
Does anyone have a protocol for doing calcium imaging without the slice being oxygenated? i.e. a recipe for a buffered solution that do not require oxygenation during imaging?
Thanks!
The carbogen bubbling is essential for for pH buffering via the sodium bicarbonate of the ACSF. Organotypic brain slice cultures and primary cultures live happily for weeks at atmospheric oxygen levels when buffered by HEPES, and the oxygen component of the carbogen may not be as important as the CO2.
HEPES is frequently used to buffer ACSF in absence of carbogen, and I think it should readily work for you without perfusion, at least for some tens of minutes and probably even longer. Pay attention that you dont have too much evaporation of ASCF during the recording, which may increase the osmolarity of the solution (basically dont have too low a volume in the imaging chamber).
Maybe you can find some HEPES based recipes here: https://www.researchgate.net/post/How_do_you_prepare_your_artificial_cerebrospinal_fluid_CSF_solution_and_what_modifications_do_you_make
Hi Jan, thanks for your answer. I will try HEPES buffered ACSF and see how it goes.
HEPES-based extracellular solution is a great suggestion. If you are not restricted by age, you could also prepare acute slices from young mice (
Hi Miaomiao, I second the HEPES suggestion for pH.
For oxygenation, you need to make sure the surface to volume ration of the solution is high (so a lot of O2 can diffuse in). Also, you may want to flush the area with 100% oxygen. ACSF flow rates used in most studies (~2 ml/min) coupled with O2 permeable tubing (non-teflon) generally lead to low levels of oxygenation anyway so your result may be comparable to perfused ones (we always use at least 6 ml/min and teflon tubing, see this study for details http://www.ncbi.nlm.nih.gov/pubmed/19200237).
Another issue to keep in mind that because you have a limited volume, substances released by the slice (e.g. synaptically and glutamate from dying cells) will not be washed out and may build up and affect the state of the slice. To counteract this, you need bigger volumes of ACSF (but again, you need to counterbalance this with the need for a high surface to volume ratio for oxygenation).
But really your best option is to perfuse. It really shouldn't be that difficult to mount a slice holder with an inlet and an outlet, some tubes, 2 bottles and a pump / gravity feed, vacuum suction (or pump if use that for feed) and a carbon canister on the setup. You will save yourself a lot of trouble with referees.
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