ICC on NF155 transfected HEKs on Multiple Sclerosis patients (samples 261-277)

OBJECTIVES

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

SAMPLES

1. 261
2. 262
3. 263
4. 264
5. 265
6. 266
7. 267
8. 268
9. 269
10. 270
11. 271
12. 272
13. 273
14. 274
15. 275
16. 276
17. 277
18. Negative control
19. 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. Negative.

RESULTS

1. Negative.
2. Negative.
3. Negative. Serum showed a nuclear pattern.
4. Negative. Serum showed a dot-like pattern.
5. Negative.
6. Negative.
7. Negative.
8. Negative.
9. Negative. Serum showed concentrated clusters of small dots outside of the nucleus.
10. Negative.
11. Negative. Serum showed a nuclear pattern.
12. Negative.
13. Negative.
14. Negative.
15. Negative.
16. Negative.
17. Negative.
18. Negative.
19. Positive.