I am trying to image Calcium with a fluorescence microscope in slices from mice injected with a virus to express GCaMP6. I would like to see spontaneous activity like in this video: https://www.youtube.com/watch?v=QXA0-MzsSWc
Currently, I have been using high potassium ACSF (100mM). I can see an overall increase of the fluorescence signal but not spontaneous blinking activity. My question is: what is a good concentration for PFC brain slices (~200um thick)? I can also use NMDAR but I do not know how much.
My research is focused on optics rather than bio so please try provide as many details as possible :)
Always put your cells or neurons or live brain or tissue slices in extracellular solution, like Ringers buffer. You should not be surprised to see a flat line if put your cells in high potassium - your neurons are continuously depolarized. Read Physiology 101, Chapter one.
You might try an "active slice" ACSF, as in this paper: https://www.nature.com/articles/nn1351#Sec8.
In their modified ACSF, KCl is 3.5 mM, MgCl2 0.5 mM, and CaCl2 1 mM.
100 mM k is too high. You are likely killing your cells if you keep them in it for too long (a brief application of 20 sec may be fine).
Too much [K]out and you are likely to saturate your signal as David said. Try from Low (2 - 3 mM) to say 10 - 15 mM for brief periods of time (1 s or so), then increase either the [K]out or the amount of time of K perfusion. You can also try synaptic stimulation with Glutamate, again, brief periods of time, if not, you might fry your cells. Play with different amounts of Glutamate (0.1 to 1 mM) should suffice (I forgot how much we used). A good advice here is to have a good perfusion system to quickly saturate your chamber and to quickly get rid of your depolarizing/exciting compounds. They cost a bit but are worth it. Good luck!
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