20170904: CASPR-2 IgG ICC IN PARANEOPLASTIC NEUROPATHIES (AMC; 15-107)

CASPR-2 IgG ICC IN HEK 293 TRANSFECTED CELLS IN PARANEOPLASTIC NEUROPATHIES (AMC; 15-107)

Objectives

To confirm sera IgG positivity towards human CASPR2  in HEK 293 transfected cells in patient AMC and to take pictures of her reactivity.

Patients

• AMC (15-107)–> the sample will be both tested in CASPR2 transfected and on-transfected cells to have a proper negative control*
• ct + 15-199 (Francisca Pedraza Gómez)

*No negative serum could be tested in CASPR2-transfected cells due to a lack of transfected coverslides.

Materials

• Human  CASPR-2-transfected HEKs 293 grown in coverslips. Fixed with PFA 4%, permeabilized and blocked with 5% goat serum in  PBS.
• Human  non-transfected HEKs 293 grown in coverslips. Fixed with PFA 4% and blocked with 5% goat serum in  PBS.
• PBS
• Blocking: 5% goat serum in  PBS
• Patient’s sera diluted 1/40 with5% goat serum in  PBS
• Primary Ab: Rabbit CASPR-2 (33994 ) (1/500)
• Secondary Abs: GAR 488 (1/1000)+ GAH 594 (1/1000)
• Vectashield mounting medium

Methods

1. Thaw CASPR2 transfected/non-transfected HEKS grown in coverslips with a little 5% goat serum in PBS.
2. Perform ICC:
   1. Add patient’s sera 1/40 1h at RT.
   2. Wash 3 times with PBS
   3. Add primary Ab 1/500 and incubate 1h at RT.
   4. Wash 3 times with PBS
   5. Add secondary Abs 1/1000 each  and incubate 1h at RT.
3. Wash 3 times with PBS
4. Mount with Vectashield mounting medium

Results

As expected patient AMC is positive for CASPR2 and bears no unspecific staining towards non-transfected HEK cells (pictures taken). Positive ct 15-199 was positive as well, vouching for the quality of the ICC.