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
