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
>> 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%
>> 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.