|Biocytin filled cortical neurons (source)|
To determine the activity of a neuron, you have to use electrophysiology to record its electrical activity. The biocytin filling method makes use of the same patch clamp electrode to record the electrical activity of the neuron and to fill it with the biocytin molecule that can be later dyed. So this method is perfect for building a neuron because with it you can correlate the shape of the neuron directly with its activity patterns.
|Neural activity correlated with neural morphology (source)|
A recent Nature Protocols paper by Marx et al. (2012) provides step by step details for how to fill and dye a neuron using the biocytin method.
The basic biocytin staining protocol is as follows:
1. make brain slices
2. fill the neuron with biocytin while recording its electrical activity
3. fix the brain slice in paraformaldehyde
4. quench the endogenous peroxidase
5. connect the biocytin to avidin (using the vectastain ABC kit)
6. colorize the avidin (using DAB and nickel)
7. mount the slices on gelatin subbed slides
8. dehydrate the slices SLOWLY through very small steps of ethanol concentration
9. clear with xylene and coverslip
Marx et al. provide some excellent specifics in the paper that make the whole process understandable and more importantly, doable. They even have a troubleshooting section which explains what might have gone wrong under several conditions.
|Marx et al., 2012 Figure 2|
So there you have it, Step 1 of neuron building. Step 2
And all the "build a neuron" steps will be indexed here.
Marx M, Günter RH, Hucko W, Radnikow G, & Feldmeyer D (2012). Improved biocytin labeling and neuronal 3D reconstruction. Nature protocols, 7 (2), 394-407 PMID: 22301777