OBJECTIVE: To test the expression of PLEXIN-D1 in NON-TRANSFECTED NEUROBLASTOMA SH-SY5Y CELLS
18.12.10
COATING:
- Prepare 1 plates of 60mm of diameter with 14 coverslips of 12mm.
- Treatment with genlatine 0.15%, incubate for 15 min
- Wash with PBS
- Trypsin-EDTA a plate of SH-SY5Y CELLS
- For A plate of 60mm: 5ml of proliferation medium + the volume that correspond to 20.000 cells/cm2
- Incubate at 37ºC for 24h.
18.12.11:
- Fix with PFA 4%. Incubation at RT during 15 min.
- Wash with PBS 1x.
- Blocking solution: Rabbit 1/40 in PBS for 1h at RT.
ICC:
- Non-transfeccted SH-SY5Y CELLS
- Serum from a healthy control diluted 1/100 in blocking solution
- Primary antibody anti-PLEXIN-D1 diluted 1/500 (ab28762, Goat polyclonal).
- Secondary antibodY: RAG488 (1/1000), RAH594 (1/1000)
- Vectashield mounting medium
RESULTS:
Postive membrane stainning pattern but could be inespecific
