Immunostaining of Human Fetal Central Nervous System Cells With the Anti-CNTF pAb
Fix human fetal CNS cells on coverslips for 10 minutes in 4% paraformaldehyde.
Wash 2 times with PBS.
Permeabilize for 10 minutes in PBS + 0.1% Triton®X-100.
Wash 3 times with PBS.
Block for 30 minutes with PBS + 3% goat serum.
Incubate in Anti-Rat CNTF pAb (5-10µg/ml) diluted in PBS + 3% goat serum for 60 minutes.
For a control, use Chicken IgY, Control Immunoglobulin (Promega
Cat. #G1161)
Wash 3 times with PBS.
Incubate with a donkey anti-chicken Cy3™ conjugated secondary antibody
(Jackson
Immunoresearch Cat.#715-165-155) diluted 1:500 for 30 minutes. Do not
expose slides to light once the Cy3™ conjugate has been applied.
In the dark, wash 3 times with PBS.
Mount with Vectashield Antifade + DAPI (Vector Labs Cat.# H1200).
