Triton X-100 BSA 20° C methanol
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
DAB - chromgen
Leitz or Olympus optical microscope
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.
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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