20170112: CNTN-1 IgG ICC in paraneoplastic neuropathies

CNTN-1 IgG ICC IN HEK 293 TRANSFECTED CELLS IN PARANEOPLASTIC NEUROPATHIES

Objectives

To test sera IgG positivity towards human CNTN1 in HEK 293 transfected cells.

Materials

  • Human CNTN-1-transfected HEKs 293 grown in coverslips. Fixed with PFA 4% and blocked with RABBIT serum 1/40 in PBS.
  • PBS
  • Blocking: RABBIT 1/40 in PBS
  • Patient’s sera diluted 1/100 with RABBIT 1/40 in PBS
  • Primary Ab: Goat CNTN-1 (AF904 ) (1/1000)
  • Secondary Abs: RAG 488 (1/1000)+ RAH 594 (1/1000)
  • Vectashield mounting medium

Patients

  • A1) 95-43
  • A2)94-292
  • A3) 97-543
  • A4) 93-40
  • A5) 92-147
  • A6) 94-55
  • B1) 93-38
  • B2) 92-133
  • B3) 01-848
  • B4) 00-1289
  • B5) 98-14
  • B6) 03-1167
  • C1) 04-2
  • C2) 13-1127
  • C3) 15-2420
  • C4) 15-1131
  • C5) 11-1167
  • C6) 00-307
  • D1) 15-2373
  • D2) 08-530
  • D3) 00-1248
  • D4) 90-92
  • D5) 04-726
  • D6) 92-38
  • E1) 98-164
  • E2) 03-838
  • E3) 05-409
  • E4) 04-1323
  • E5) 16-3512
  • E6) 01-203
  • F1) 93-94
  • F2) 97-506
  • F3) 02-616
  • ct hvh1

Methods

  1. Transfected HEKS grown in coverslips were fixed and blocked with 1/40 Rabbit serum and frozen at -80ºC.
  2. Thaw these cells with a little 1/40 Rabbit serum 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

Positive control HVH1 (which has been analyzed in the second sample batch of this experiment) did not stain CNTN1. Since proper HEK transfection had been previously demonstrated and there’s no point in doubting the antibodies (all of them have performed as per usual in batch 1), most likely some sort of pipetting mistake must have happened. As a result, the experiment must be discarded. Samples will be  tested in forthcoming experiments.

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