ICC on NF155 transfected HEKs on Multiple Sclerosis patients (samples 239-260)

OBJECTIVES

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

SAMPLES

1. 239
2. 240
3. 241
4. 242
5. 243
6. 244
7. 245
8. 246
9. 247
10. 248
11. 249
12. 250
13. 251
14. 252
15. 253
16. 254
17. 255
18. 256
19. 257
20. 258
21. 259
22. 260
23. Negative control
24. 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.
4. Negative. Serum marked a nuclear pattern.
5. Negative.
6. Negative.
7. Negative.
8. Negative.
9. Negative.
10. Negative.
11. Negative.
12. Negative.
13. Negative. Serum marked a dot-like pattern in the cytoplasm.
14. Negative.
15. Negative. Serum marked a strong dot-like pattern in the cytoplasm.
16. Negative.
17. Negative.
18. Negative. Serum marked a nucleolar pattern.
19. Negative.
20. Negative. Serum marked a dot-like pattern in the cytoplasm.
21. Negative.
22. Negative.
23. Negative.
24. Positive.