Wash cultured hippocampal neurons with pre-warmed tyrode.

Wash cultured hippocampal neurons with pre-warmed tyrode. (1/3)

Wash cultured hippocampal neurons with pre-warmed tyrode. (2/3)

Wash cultured hippocampal neurons with pre-warmed tyrode. (3/3)

Fix the cells with fixative solution for 0h 15m 0s at Room temperature.

After fixation, wash the cells with PBS.

Wash the cells with PBS. (1/3)

Wash the cells with PBS. (2/3)

Wash the cells with PBS. (3/3)

Incubate with blocking buffer for 0h 30m 0s atRoom temperature.

Wash the cells briefly with PBS and incubate the cells with primary antibodies (1:500 ~ 1:2000) in a blocking buffer for 1h 0m 0s at Room temperature on a rocking platform.

Wash with PBS on a rocking platform.

Wash with PBS for 0h 2m 0s on a rocking platform. (1/5)

Wash with PBS for 0h 2m 0s on a rocking platform. (2/5)

Wash with PBS for 0h 2m 0s on a rocking platform. (3/5)

Wash with PBS for 0h 2m 0s on a rocking platform. (4/5)

Wash with PBS for 0h 2m 0s on a rocking platform. (5/5)

Incubate the cells with secondary antibodies (for example Alexa-fluor-labeled) (1:1000) in a blocking buffer for 0h 45m 0s on a rocking platform atRoom temperature in the dark.

Decant the secondary antibodies and wash with PBS.

Wash with PBS for 0h 2m 0s each in the dark. (1/5)

Wash with PBS for 0h 2m 0s each in the dark. (2/5)

Wash with PBS for 0h 2m 0s each in the dark. (3/5)

Wash with PBS for 0h 2m 0s each in the dark. (4/5)

Wash with PBS for 0h 2m 0s each in the dark. (5/5)

Observe the fluorescence signal using an inverted confocal microscope or mount the samples with Prolong Gold antifade reagent for long-term storage