Immunocytochemistry of primary neurons
giulia.tombesi
Abstract
This protocol describes the steps to obtain a great staining of primary neurons.
Steps
Cells Fixation
Wash cells with PBS1X/HBSS
Fix cells with paraformaldehyde (PFA) 4% for 0h 30m 0s at room temperature
Wash cells with PBS1X for 0h 5m 0s for three times at room temperature
Cells permeabilization and saturation
Permeabilize cells with 0.3% Triton X-100 (in PBS1X) for 0h 5m 0s at room temperature
Wash cells with PBS1X for 0h 5m 0s for three times at room temperature
Saturate cells in blocking solution for at least 0h 30m 0s
Wash cells with PBS1X for 0h 5m 0s for three times at room temperature
Incubation with primary antibody
Incubate with primary antibody in working solution for 1h 30m 0s at room temperature
Wash cells in working solution for 0h 5m 0s for three times at room temperature
Incubation with secondary antibody
Incubate with secondary antibody in working solution for 1h 0m 0s at room temperature in the dark
Wash cells in working solution for 0h 5m 0s for three times at room temperature
Wash cells in PBS1X for 0h 1m 0s for three times at room temperature
Mounting
Rinse coverslips in dH2O
Mount the coverslips on slides with mounting medium
