[Neuroscience] Biocytin fills?

Imre Vida via neur-sci%40net.bio.net (by Imre.Vida At anat.uni-freiburg.de)
Thu Nov 16 14:25:00 EST 2006


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



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