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Paraformaldehyde/glutaraldehyde fixation(method of choice for double labeling of membrane bound and cytoskeletal antigens)
- Rinse cells with PBS at room temperature.
- Fix in 3% paraformaldehyde, 0.02% glutaraldehyde in PBS for 15 minutes at room temperature.
- Permeabilize by immersing cells for 10 seconds in 100% methanol at -20°C.
- Incubate cells for 10 minutes in 0.5 mg/mL NaBH4 in PBS, pH 8.0, to reduce aldehyde groups and then repeat 3 times.
- Rinse cells with PBS.