20170213: IgG primary culture Schwann cell ICC in paraneoplastic neuropathies

Primary culture  Schwann cell IgG ICC is performed to test paraneoplastic neuropathy patients’ staining pattern.

PATIENTS

The following patients  were analyzed by IgG:

• 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

Materials

• Rat primary culture of Schwann  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
• Secondary Abs: GAH 488 IgG (1/1000) (box 25)
• Vectashield mounting medium

RAT PRIMARY CULTURE SCHWANN CELL ICC 

1. Primary culture Schwann cells  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 secondary Ab GAH 488 IgG (1/1000) each and incubate 1h at RT.
4. Wash 3 times with PBS.
5. Mount with Vectashield mounting medium.

RESULTS

Edited 20170228: a loss of fluoresecence intensity is suspected due to discrepancies between the results observed by two independent viewers in two different days. As a result, the experiment will be repeated.