ICC on NF155 transfected HEKs on Multiple Sclerosis patients (samples 278-327 and 383-386)

OBJECTIVES

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

SAMPLES

  1. 278
  2. 279
  3. 281
  4. 283
  5. 288
  6. 289
  7. 290
  8. 291
  9. 292
  10. 293
  11. 294
  12. 295
  13. 297
  14. 298
  15. 299
  16. 300
  17. 301
  18. 302
  19. 305
  20. 306
  21. 307
  22. 309
  23. 311
  24. 312
  25. 315
  26. 318
  27. 322
  28. 323
  29. 324
  30. 325
  31. 326
  32. 327
  33. 383
  34. 385
  35. 386 (was a mislabeled CSF sample)
  36. Negative control
  37. 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. Serum showed nuclear pattern.
  3. Negative. Serum showed a faint nuclear pattern.
  4. Negative. Serum showed irregular nuclear pattern.
  5. Negative.
  6. Negative. Serum showed an irregular pattern.
  7. Negative.
  8. Negative.
  9. Negative.
  10. Negative. Serum showed nuclear pattern.
  11. Negative.
  12. Negative. Serum showed an irregular pattern.
  13. Was not visible due to error in the procedure.
  14. Negative. Serum showed nuclear pattern.
  15. Negative.
  16. Negative.
  17. Negative.
  18. Negative.
  19. Negative. Serum showed dot-like pattern.
  20. Negative.
  21. Negative. Serum showed irregular nuclear pattern.
  22. Negative. Serum showed an irregular pattern.
  23. Negative.
  24. Negative.
  25. Negative.
  26. Negative.
  27. Negative.
  28. Negative.
  29. Negative.
  30. Negative.
  31. Negative.
  32. Negative. Serum showed a nuclear pattern.
  33. Negative.
  34. Negative. Serum showed a nuclear pattern.
  35. Negative. Was a mislabeled CSF sample.
  36. Negative.
  37. Positive.



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