ICC on NF155 transfected HEKs on Multiple Sclerosis patients (samples 101-106)

OBJECTIVES

To determine the existence of anti-NF155 antibodies in a subset MS patients.

SAMPLES

1. 101
2. 102
3. 103
4. 104
5. 105
6. 106
7. Negative control (Edu serum)
8. Positive control (15-195)

MATERIALS

• Human NF155-transfected HEKs 293 grown in coverslips. Fixed with PFA 4% and blocked with GOAT serum 5% in PBS.
• PBS.
• Blocking: GOAT 5% in PBS.
• Patient’s sera diluted 1/100 with GOAT 5% in PBS.
• Primary Ab: Chicken anti-NF155: AF3235 (1/1000).
• Secondary Abs: GAH 594 (1/1000) + GAC 488 (1/1000).
• Vectashield mounting medium.

PROCEDURE

• NF155-transfected HEKs were grown and fixed on coverslips and frozen at -80ºC. They where thawed before the ICC with Goat Serum 5%.
• Added patient’s sera at 1/100 dilution in Goat Serum 5% and incubated 1h at RT.
• Washed 3 times with water.
• Added primary commercial anti-NF155 Ab: AF3235 at 1/1000 dilution and incubate 1h at RT.
• Washed 3 times with water.
• Added secondary Abs: GAH 594 and GAC 488 1/1000 each and incubated 1h at RT.
• Washed 3 times with water.
• Added Vectashield mounting medium and put onto microscope slides.
• The slides were observed with a fluorescense microscope to determine wether or not the patients’ sera presented anti-NF155 antibodies.

RESULTS

1. Negative.
2. Negative.
3. Negative.
4. Negative.
5. Negative.
6. Negative.
7. Negative.
8. Positive.