Hi again:
I am trying to optimize a protocol for imaging calcium dynamics in a cell population in the dentate gyrus and am facing some trouble. I made a similar question and got 1 answer but I guess a new one attracts more people and I am going to be more specific.
I am making coronal acute slices and staining them with a calcium indicator, but when I mount my slice I don't manage to focus it under the 40x objective or sometimes even the 20x one. I place my slice onto a glass coverslip that fits into a chamber where ACSF flows through.
I think the problem is the dentate gyrus detaches from the coverslip, maybe the orientation makes it easy. I have tried attaching the slice with PEI and poly-L-lysine (PLL, coating the coverglass over night) and putting a small piece of metal on the slice but it is not working for me. I have read that PLL doesn't work so well with tissue and gelatin sticks better, but I am also afraid that my slice can wrinkle and get stuck too soon. Any ideas?
Thanks in advance and kind regards,
Roberto
Dear Roberto,
Classically(patch clamp and calcium imaging) we used platinum harp which is heavy enough to stick the slice on the bottom of the chamber, but which is not ionizable. THere are many commercially avaible harps that perfectly fit in the recording chamber. However, you can also make it yourself.
Pascal
Yes, I think that will be my final choice. Thanks for your reply
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