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[Neuroscience] Biocytin labeling in adult brain slices

Hyunchul Lee via neur-sci%40net.bio.net (by hlee from medsci.usyd.edu.au)
Wed Mar 14 17:42:41 EST 2007


Hi,

I'm not sure how much experience or prior success you had with the 
technique, so I'll tell you what I know...
After a recording, did you fill the cell by iontophoresis?
Biocytin doesn't diffuse as easily as say neurobiotin, we use 0.5~1% 
neurobiotin in our solutions and pulse positive current for around 10 
minutes before coming off the cell.
Sometimes, when coming off the cell, you may be pulling the cell out of 
the slice too, so you need to come off gently.
Another possible problem might be when and if you're resectioning, the 
filled cell might be cut off.
This is all assuming there's no problem with your ABC/DAB protocol.

Now with gliosis... I'm not totally sure, but might there not be the 
possibility that some of your cells get eaten up between when you 
recorded and when you fixed your tissue?  Unless it was fixed 
immediately..?  Just guessing.

Hope I made a contribution..
Hyunchul

Karthik Rajasekaran wrote:
> Hi
> I have only limited success (approx. 50%) with biocytin labelling of neurons 
> from which I record whole cell currents from thalamic relay neurons. The 
> protocol that I use is fairly standard in which singal is amplified by 
> incubating the sections in ABC and then reacted with DAB. The slices are 
> obtained 8-9 month old rats, and often those that present with extensive 
> gliosis....though I do not see any reason why that would affect it as long 
> as I get my cell to record...  Any advice / suggestions will be greatly 
> appreciated. Thank you!
> Karthik
>
>
>
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