ICC on Neuroblastoma Cells using MS serum

OBJECTIVES

To screen for reactivity against neurons on Multiple Sclerosis patients.

MATERIALS

• Human differentiated neuroblastoma cells grown in coverslips.
• PBS.
• Blocking: GOAT 5% in PBS.
• Patient’s sera diluted 1/100 with GOAT 5% in PBS.
• Primary Ab: AF3235 (1/200 dilution).
• Secondary Abs: GAH 488 (1/1000) + GAC 594 (1/1000).
• Vectashield mounting medium.

PROCEDURE

• The cells were grown and differentiated using this protocol.
• Patient’s sera were diluted 1/100, and the coverslips were placed in a contained humidity box with parafilm, with the cells above and placing the 30microL drops carefully on top of the coverslips.
• 1h incubation.
• Wash 3 times with PBS1x.
• The primary antibody (AF3235 1/200 dilution) was placed in 30microL drops on top of the coverslips as before.
• 1h incubation
• Wash 3 times with PBS1x.
• The secondary antibody: (GAH488 and GAC594 diluted 1/750) was placed in 30microL drops on top of the coverslips as before.
• 1h incubation.
• Wash 6 times with PBS1x.
• Added Vectashield mounting medium and put onto microscope slides.
• The slides were observed with a fluorescense microscope.

RESULTS

The results are judged in a scale of 0 to 3 +