Objective: to test IgG reactivity against SH-SY5Y neuroblastoma derived neurons in patients iwht small fiber neuropathy
ICC protocol:
- Fix diferenciated SH-SY5Y cells with 4% PFA 5 min
- Wash 2 times with PBS 1x
- Block with goat serum 5% for 1h at RT
- Dilute serum 1/100 in blocking solution, incubate for 1h
- Wash with PBS 1x
- Commercial anti-NF Ab 1/200 diluted in blocking solution, incubate for 1h
- Wash with PBS 1x
- Secondary antibodies: GAH 488 (1/500), GAC 594 (1/500)
- Wash with PBS 1x (not water)
- Vectashield mounting medium
Results: see table and photos
samples 1++, 2+, 33++, 45+ were positive (axonal stainning pattern)
| IVIG SAMPLE | RESULT |
|---|---|
| 1 | POSITIVE ++ |
| 2 | POSTIVE + |
| 3 | negative |
| 4 | negative |
| 5 | negative |
| 6 | negative |
| 7 | negative |
| 8 | negative |
| 9 | negative |
| 10 | negative |
| 11 | negative |
| 12 | negative |
| 13 | negative |
| 14 | negative |
| 15 | negative |
| 16 | negative |
| 17 | negative |
| 18 | negative |
| 19 | negative |
| 20 | negative |
| 21 | negative |
| 22 | negative |
| 23 | negative (somas) |
| 24 | negative (somas) |
| 25 | negative (somas) |
| 26 | negative |
| 27 | negative |
| 28 | negative (somas) |
| 29 | negative |
| 30 | negative |
| 31 | negative |
| 32 | negative |
| 33 | POSITIVE ++ (somas, axon, nuclei) |
| 34 | repeat |
| 35 | negative |
| 36 | negative |
| 37 | negative |
| 38 | negative |
| 39 | negative |
| 40 | negative |
| 41 | negative |
| 42 | negative |
| 43 | negative (doubts, repeat) |
| 44 | negative |
| 45 | POSITIVE + (somas, axon) |
| 46 | negative |
| 47 | negative (somas) |
| 48 | negative (somas) |
| 49 | negative |
| 50 | negative |
| 51 | negative (nucleos) |
| 52 | negative |
| 53 | negative (somas) |
| 54 | negative (somas) |
| 55 | negative |
| 56 | negative |
| 57 | negative |
| 58 | negative |
| 59 | negative (somas) |
| 60 | negative (somas) |
| CNTN1+ | POSITIVE |
| Healthy donor | negative |
| IVIG SAMPLE | RESULT |
|---|---|
| 1 | POSITIVE ++ |
| 2 | POSITIVE (MILD) |
| 17 | negative |
| 28 | negative |
| 33 | POSITIVE: nuclei and axons |
| 34 | negative |
| 43 | negative |
| 45 | POSITIVE |
| CNTN1+ | POSITIVE |
| Healthy donor | negative |
