[Neuroscience] Re: Biocytin fills?

Bill via neur-sci%40net.bio.net (by connelly.bill At gmail.com)
Wed Nov 29 23:08:37 EST 2006


Excellent! Thank you very much.

Couple of quesions: When you say form an outside-out patch, do you
simpley mean withdraw the electrode from the cell? Is there any
particular method to this? Fast, slow etc...?

The 4% PFA, make that in distilled water, or 0.1MPB?

Again, thankyou very much!


Imre Vida wrote:

> Hi Bill,
>
>
> > I see a number of unanswered posts on this subject; but does anyone
> > have a method for biocytin fills of neurons recorded by whole-cell
> > patch-clamp in brain slices? Preferably one that doesn't require
> > resectioning?
> >
> we use 0.05 - 0.2 % ( 0.5 - 2 mg/ml) biocytin in the pipette solution.
> KGluconate based solutions work very well, KCl is more problematic for
> labeling.
>
> Obtain an out-side-out patch when you finish recording, and fix the
> slice asap, but not before 20-30 min from the start of the recording
> (time for labeling distal axons collaterals).
> Fixative: 4% PFA for LM is fine, for EM you need GA also. Overnight.
>
> For visualization you don't need to reslice the sections,  if you aim
> for LM investigations only.
>
> Main steps:
> - wash out fixative
>   rinse several times in 0.1M PB
>
> - block endogenous peroxidase activity
>   10-15' 1% H2O2
>   wash out H2O2 thoroughly   5-6 x 10-15' PB
>
> - enhance penetration
>   for LM only treatment with a detergent should be OK (0.1-0.3%
>   Trinton-X)
>
> For brightfield LM
> - incubate in avidin-peroxidase complex (1:100 dilution Vector Elite kit)
>   overnight, 4 C, shaker
>   wash thoroughly  5-6 x 10-15' PB
>
> - preincubate in DAB (3,3'-Diaminobenzidine) 0.5 mg /ml in PB   ~20 min
>   (CAVE: DAB is a likely carcinogen!)
>   add H2O2   1% 10 ul/ml
>   check development of the reaction under the microscope
>   cell should be visualized and darkly stained in a few minutes
>   wash thoroughly
>   (DAB can be neutralized by NaClO solution)
>
>
> For fluorescent LM
> - incubate in avidin-conjugated fluorochrome overnight
>   (we usually use Alexa dyes from Molecular Probes at a dilution of 1:500)
>   wash thoroughly
>
>
> For LM you can directly embed the slices in aqueous mounting medium.
> 
> 
> best 
> 
> imre



More information about the Neur-sci mailing list