ICC on NF155 transfected HEKs on Multiple Sclerosis patients (samples 217-228)

OBJECTIVES

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

SAMPLES

1. 217

2. 218

3. 220

4. 221

5. 222

6. 223

7. 224

8. 225

9. 226

10. 227

11. 228

12. Negative control (Edu serum)

13. 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 incubated 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. Marked nuclear pattern.

6. Negative. Small dot-like pattern.

7. Negative. Marked nuclear pattern.

8. Negative.

9. Negative. Faint nuclear pattern.

10. Negative.

11. Negative. Marked nuclear pattern.

12. Negative.

13. Positive.