實驗概要
The following protocol provides a method of primary cultures for IHC – viability assays.
實驗步驟
1. Preparation of primary mesencephalic cultures
1) The mesencephalic neurons and glia are dissociated from neuronal tissue with trypsin (final concentration, 26 mg/ml in 0.9% [w/v] NaCl)
2) Cells are plated on coverslips previously treated with poly-L-lysine (5 mg/ml).
3) The media consists of DMEM, 10% (v/v) FBS, 10% (v/v) HS, penicillin (100 U/ml), and streptomycin (100 mg/ml).
4) Four days later, the cells were treated for 48 hr with AraC (20 mM) to inhibit the growth of glial cells.
2. Lentiviral transductions of primary cultures
1) AraC-treated primary cultures are transduced with lentiviral particles in the presence of polybrene (6 mg/ml).
2) Control cells were incubated without lentivirus.
3) After a 72 hr transduction period, the cells were treated with fresh media for an additional 48 hr and analyzed immunocytochemically.
3. Immunocytochemistry of primary midbrain cultures
1) Primary cells were fixed in 4% (w/v) paraformaldehyde in PBS for 30 min.
2) The cells were permeabilized and blocked simultaneously for 1 hr with PBS containing 1% (w/v) BSA, 10% (v/v) FBS, and 0.3% (v/v) Triton X-100.
3) After washing with PBS, the cells were treated overnight at 4°C with an anti-MAP2 monoclonal IgG (1:500) and an anti-TH polyclonal antibody (1:500) to monitor relative dopaminergic cell viability.
4) To determine the lentiviral transduction efficiency, untransduced cells or cells transduced with lacZ lentivirus (expressing beta-galactosidase fused to the V5 epitope) are treated at room temperature for 1 hr with:
a. an anti-V5 monoclonal antibody (1:200) and an anti-MAP2 polyclonal antibody (1:500), or
b. an anti-V5 monoclonal antibody (1:200) and an anti-TH polyclonal antibody (1:500).
5) After washing, the cells were treated for 1 hr at room temperature with goat anti-mouse IgG conjugated to AlexaFluor? 488 (1:1000) and goat anti-rabbit IgG conjugated to AlexaFluor? 594 (1:2000).
6) The primary and secondary antibodies were prepared by diluting stock antibody solutions in PBS with 1% (w/v) BSA.
7) The coverslips were mounted onto slides using ProLong Gold Antifade? reagent, dried at room temperature overnight, and sealed with clear nail polish.
4. Measurement of primary neuron viability
1) MAP2- and TH-immunoreactive primary neurons can be counted in 10 randomly chosen observation fields for each experimental condition at a total magnification of 200x.
2) The data should be expressed as the percentage of MAP2-positive neurons that are also TH-positive.
3) Each experiment should be repeated 3 to 4 times using embryonic neurons isolated from independent animals.
4) Statistical analyses can be carried out using the program GraphPad Prism?, Version 4.0
