OBJECTIVE: To test the expression of PLEXIN-D1 in HEK 293 cells
Day 1:
- Prepare one plates of 60mm of diameter with 14 coverslips of 12mm.
- Treatment with Poly-D-Lysine (diluted 1/40 with boratte buffer)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS
- Trypsin-EDTA a plate of HEK293.
- For every plate of 60mm: 5ml of HEK medium + the volume that correspond to 1.200.000 of cells.
- Incubate at 37ºC for 24h.
Day 2:
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Blocking solution: Rabbit serum 1:40 in PBS for 1h at RT.
- Remove the blocking solution and freeze at -80ºC
ICC:
- Primary antibody anti-PLEXIN-D1 diluted 1/50 (ab28762, Goat polyclonal).
- Secondary antibodY: RAG488 (1/1000).
- Vectashield mounting medium
RESULTS: Postive membrane stainning pattern compatible with expression of plexin-D1
