Pbs

Triton X-100 BSA 20° C methanol

2% paraformaldehyde

Antibodies to:

Neurofilament 200 (NF200), 68 (NF68), monoclonal antibodies from Amersham, Australia

Cyclic GMP protein kinase, polyclonal antibody, a kind gift from Professor P. Greengard, Rockefeller University

Parvalbumin, MAP 2, calbindin D-28, glutamate, monoclonal antibodies from Sigma, Chemical Co.

Neuron-specific enolase (NSE), polyclonal antibody from Sigma Chemical Co.

Thy-1 (French and Jeffrey, 1986)

Alkaline phosphatase, horseradish peroxidase, FITC and RITC-conjugated secondary antibodies from Jackson ImmunoResearch Laboratories or BioRad Laboratories

NBT/BCIP

DAB - chromgen

Leitz or Olympus optical microscope

Goat serum

2. Neuronal Characterization

Purkinje neurons were identified by markers and morphological characteristics following days in vitro culture. Coverslips were fixed in either —20° C methanol for 30 min prior to immunostaining for neurofilament 200 (NF200 1/100), cyclic GMP protein kinase (G-kinase 1/1000), parvalbumin (1/250), MAP2 (1/100), and neurofilament 68 (NF68 1/50) or in 2% paraformaldehyde in PBS at room temperature for 30 min prior to immunostaining for calbindin D-28 (1/100), NSE (1/100), amino acid glutamate (1/100), GABA (1/100), and Thy-1 (1/100) (French and Jeffrey, 1986).

1. Following fixation, wash three times with PBS.

3. Wash three times with PBS.

4. Partially permeabilize with 0.5% Triton X-100 in PBS for 2min.

5. Add primary antibodies in dilutions determined specifically for the antibody source. Sample dilutions from this laboratory were given earlier.

6. Visualization is by enzyme-linked antibodies, either alkaline phosphatase or horseradish peroxidase with NBT/BCIP or DAB chromogen, respectively.

7. View under a high-quality optical microscope.

Double fluorescent staining, to identify Purkinje neurons, was performed using rabbit antibody to NSE, a neuronal marker, and a mouse monoclonal antibody to calbindin D-28, a Purkinje neuron marker, for 1 h at room temperature or

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