Okay, I see...
In our experience, filling cells by diffusion alone hasn't been very
reliable. I thoroughly recommend iontophoresis, even given your long
I also think neurobiotin does fill cells better, and is easier to
prepare too. We don't even bother to vortex our intracellular solution
after adding neurobiotin, since it dissolves quite quickly on its own.
As for how long you wait till fixation... normally we wait ~30 minutes
just so that any extracellular biocytin/neurobiotin may be metabolized
and hence reduce noise from any leaks. You can leave it for an hour or
two... in theory it should last for at least 24 hours intracellularly
(though I haven't tried that). However, I was thinking more about what
you said about your tissue being gliotic..
Karthik Rajasekaran wrote:
> Hi Hyunchul and Bill:
> Thank you for your inputs. The ABC/DAB works fine, and cells that
> stain, stain very well and very nicely. I do not iontophorise it; but
> let passive diffusion occur through the patch pipette, as my
> recordings are usually for about an hr... Also, I do not resection my
> 300 micron thick slices. The way I do it is that after recording, I
> give a test pulse and slowly withdraw the pipette so that I would know
> that its coming off the cell slowly enough. After that I place it in a
> petri dish and leave it for considerable time ... 45 mins at 29 degree
> C (why so?? cannot really say that it definitely does... just
> superstitious because that helped it work many times earlier in our
> hands, and so it became a "standard"). Do you fix immediately? And how
> long do you record... rather what is the shortest recording time with
> which you have been able to obtain good labeling?
>> But, I think that I will try Neurobiotin, which if it does the trick
> will be very helpful. Thanks again for your inputs. I really
> appreciate it. Cheers!
>> PS: Some jinx in my outlook express newsgroup settings and so I am
> sending this as personal replies rather than as a newsgroup post. Hope
> you guys do not mind that. Thanks.
>> ----- Original Message ----- From: "Hyunchul Lee"
> <hlee from medsci.usyd.edu.au>
> To: "Karthik Rajasekaran" <kr5z from virginia.edu>; <neur-sci from net.bio.net>
> Sent: Wednesday, March 14, 2007 6:42 PM
> Subject: Re: [Neuroscience] Biocytin labeling in adult brain slices
>>>> 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..
>>>> Karthik Rajasekaran wrote:
>>> 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!
>>> Neur-sci mailing list
>>>Neur-sci from net.bio.net>>>http://www.bio.net/biomail/listinfo/neur-sci>>>>>>>>>