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 test was performed twice, and the results will be separated in two tables. The results are judged in a scale of 0 to 3 +

On this first test the fluorescence was very strong and we suspect on further tests the more postive ones won’t be so clear.

The results of the following test are not very trustworthy, as it appears the neurons weren’t differentiated enough and in some cases were quite scarce. All of them should be repeated but the following results can serve as a guide.