20140603 ICC cntn+nfasc new patient

Objective

The aim of this experiment is to test sera from one new patient

Materials

• Transfected HEK cells with CNTN1-human/NFASC155 grown in coverslips
• Patient sera
• Antibodies:
  • mouse-anti-contactin
  • chicken-anti-neurofascin
  • GAM-488
  • GAH-594
  • GAC-488

Procedure

1. Take one coverslip per condition
2. Prepare sera 1:40 in goat serum. Add a drop (40µl) of each preparation to the parafilm. Incubate for 1h
3. Wash 3 times with PBS
4. Incubate primary antibody 1h: (1) mouse-anti-contactin 1:500 (2) chicken-anti-neurofascin 1:2000
5. Wash 3 times with PBS
6. Prepare secondary antibody: (A) GAH IgG-594 1:500 + GAM-488 1:500 (B) GAC-488 + GAH-594. Add a drop and incubate 1h.
7. Wash 3 times with H2O
8. Mount with Vectashield (with DAPI)

Results & conclusions

This patient seems positive for CNTN. We’re going to test it again with fresh HEKs transfected with CNTN(mouse).