20170117: NF140 IgG ICC in paraneoplastic neuropathies

NF-140 IgG ICC IN HEK 293 TRANSFECTED CELLS IN PARANEOPLASTIC NEUROPATHIES

Objectives

To test sera IgG positivity towards human NF-140 in HEK 293 transfected cells.

Materials

• Human NF-140-transfected HEKs 293 grown in coverslips. Fixed with PFA 4% and blocked with GOAT 5% in PBS.
• PBS
• Blocking: GOAT 5% in PBS
• Patient’s sera diluted 1/100 with GOAT 5% in PBS
• Primary Ab: Chicken NF155 (AF3235) (1/1000)
• Secondary Abs: GAC 488 (1/1000)+ GAH 594 (1/1000).
• Vectashield mounting medium

Patients

• 97-506 (NF-140)
• positive ct 16-069

Methods

1. Transfected HEKS grown in coverslips were fixed and blocked with GOAT 5% in PBS and frozen at -80ºC.
2. Thaw these cells with a little GOAT 5% in PBS
3. Perform ICC:
   1. Add patient’s sera 1/100 1h at RT.
   2. Wash 3 times with PBS.
   3. Add primary Ab 1/1000 and incubate 1h at RT.
   4. Wash 3 times with PBS.
   5. Add secondary Abs 1/1000 each and incubate 1h at RT.
4. Wash 3 times with PBS.
5. Mount with Vectashield mounting medium.

Results

Sample 97-506 (NF-140) was negative (positive control OK).