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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