2019.07.24 Morning:
- Prepare 3 plates of 60mm of diameter with 24 coverslips of 9mm.
- 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 LEC1 cells
- For every plate of 60mm: 5ml of LEC1 medium + the volume that correspond to 1.000.000 of cells.
- Incubate at 37ºC for 24h.
2019.07.25 Afternoon:
Prepare the DNA and lipofectamine:
- 3 plates: 1) CNTN1, 2) Caspr1, 3) CNTN1/Caspr1 (Same protocol as transfection in HEK cells)
- 8 µg DNA+ 250 µl Optimem / plate
- 12 µL of Lipofectamine + 250 µl Optimem / plate
- Incubate at RT for 5 min.
- Unify DNA with Lipofectamine and incubate at RT for 30 min.
- Add 500microL of the mix of DNA and Lipofectamine to the plate.
- Incubate O/N at 37ºC.
2019.07.26 Morning:
- 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 (CNTN1 and CNTN1/Caspr1), and Goat 5% for Caspr1
- Remove the blocking solution and freeze at -80ºC.
Check the transfection – ICC:
CNTN1 and CNTN1/Caspr1:
- Blocking solution: rabbit serum 1:40 in PBS
- Ac anti-CNTN1 diluted 1/1000
- RAG 488 IgG diluted 1/1000
- Vectashield mounting medium
Result: cells were well transfected with CNTN1
Caspr 1:
- Blocking solution: goat 5% in PBS
- Ac anti-Caspr1 diluted 1/250
- GAR 488 IgG diluted 1/1000
- Vectashield mounting medium
Result: cells were well transfected with Caspr1
Problems: 1.000.000 of cells. per plate were too many, because de cells grew very fast and they were 100% confluent the second day. Also, because there were too many cell, they did not have enough space and were not in the usual conformation (fusiform), and looked very rounded. After ICC, may cells had detached. Next time I will add 500.000. and try coating with laminin
