OBJECTIVES
To determine the existence of anti-NF155 antibodies in a subset MS patients.
SAMPLES
- 127
- 128
- 129
- 130
- 131
- 132
- 133
- 134
- 135
- 136
- 137
- Negative control (Edu serum)
- 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
- Negative.
- Negative.
- Negative.
- Negative.
- Negative.
- Negative.
- Negative.
- Negative.
- Negative.
- Negative.
- Negative.
- Negative.
- Positive.
Sample 1 showed a nuclear marking pattern. Samples 2 and 3 presented a marked dot-like cytoplasmic inclusions.