Troubleshooting ACSF

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Dpk
Dpk's picture
Troubleshooting ACSF

Hi,
 
     I have been trying to record field potentials from hippocampal slices (400um), but, not getting any response.  I have tried 3 different recipies for ACSF prepartion.  The slices are taken within 15 min from the time of decapitation. The slices are let to recover for 30 min before recording.  I have tried stimulating the slices (10-200uA) and Mg Free ACSF with elevated KCl (4mM)  for inducing epileptic activity. None of them seem to work. 

1. Is there anyway to confirm that ACSF am using is working?  The osmolarity is usually 308-310 and pH 7.4.

2. Is there any means of confirming tissue viablity? ( the slice looks very glittery and the edges start to fold inwards in over 2hrs)

3. I am planning to use bicuculline methiodide for inducing epilepsy in the slices. The conc i need to use is 20uM.  I have 10mg vials of bicuculline.  Can some tell me how to prepare stocks and how to use the drug?

Desperately waiting for an answer!!

Thanks in advance,
Deepak.

Hss
Hss's picture
 ...a made slices with cortex

 ...a made slices with cortex and hippocampus, and I can see the ventricles too; normaly I can see the Ependymal cells from the ventricles moving a lot and that´s my reference to know if the neurons  / slices are alive.     

The FFM
The FFM's picture
 Have you tried waiting until

 Have you tried waiting until after you have bubbled the solution with 95% oxygen, 5% CO2 to saturation before you add the CaCl2?

Dpk
Dpk's picture
Thanks for the replies.

Thanks for the replies.

@ Hss:  The sections i take are horizontal and ventral most.  I am not sure if i would get the ventricles at this level.  Am trying to get the entrorhinal cortex ,Amygdala and hippocampus in the same slice.

@ FFM: Yes. I have tried bubbling for 20 min before adding cacl2. I have also tried bubbling right from the beginning (i.e) from the deionized water.  Nothing seems to work.   How do i make sure the solution is saturated? i bubble for around 20-30 min then add CaCl2 and bubble again till i finish the recording, which is approx. 4 hrs.  

Does anyone use VF 200 vibrotome?. Can u suggest the optimum settings (oscillations and Advance) for 4-6 wk old wistar rats??  Now i use Advance:3  and oscillations: 8.

thanks.