20180219-21: COATING AND TRANSFECTION FOR CNTN1, NF186, CASPR1 and CASPR2

Day 1- 2018.02.19:

• Prepare 6 plates of 60mm of diameter with 24 coverslips of 9mm.
• Treatment with Poly-D-Lysina (diluted 1/40 with boratte buffer)
• Incubate for 1h (37ºC, 5% CO2).
• Wash with PBS 4x.
• Trypsin-EDTA a plate of HEK293.
• Manual counting: 6.55×10^6cells/mL
• For every plate of 60mm: 5ml of HEK medium + the volume that correspond to 800.000 of cells: 122microL.
• Incubate at 37ºC for 24h.

Day 2- 2018.02.20:

[CONTACTIN] =1.181microg/microL.

[NF186]=1.120microg/microL.

[CASPR1]=0.74microg/microL.

[CASPR2]=0.5microg/microL.

*For every plate of 60mm of diameter –> 8 microg of DNA + 12 microL of Lipofectamine.

• Contactin : 2 plates
  • 13.55 microL of DNA in 500microL of Optimem.
  • 24 microL of Lipo in 500microL of Optimem.
• NF186: 2 plates
  • 14.29microL of DNA in 500microL of Optimem.
  • 24 microL of Lipo in 500microL of Optimem.
• CASPR1: 1 plate
  • 10.81microL of DNA in 250microL of Optimem.
  • 12 microL of Lipo in 250microL of Optimem.
• CASPR2: 1 plate
  • 16microL of DNA in 250microL of Optimem.
  • 12 microL of Lipo in 250microL of Optimem.

– Incubate at RT for 5 min.

– Unify every DNA with the appropriate amount of Lipo and incubate at RT for 30 min.

– For every plate: 5ml of HEK medium and 500microL of every mix of DNA and Lipo.

– Incubate O/N at 37ºC.

Day 3- 2018.02.21:

• Fix with PFA 4%. Incubation at RT during 10min.
• Wash with PBS 1x.
• Blocking solution:
  • For CNTN1: Rabbit serum 1/40 in PBS for 1h at RT.
  • For NF186: 5% goat serum in PBS for 1h at RT.
  • For CASPR1: 5% goat serum in PBS for 1h at RT.
  • For CASPR2: 5% goat serum in PBS for 1h at RT.
• Remove the blocking solution and freeze at -80ºC.

Check the transfection – ICC:

For Contactin1:

• Blocking solution: Rabbit serum 1/40 in PBS
• Primary antibody: AF904 Diluted 1/1000
• Secondary antibody: RAG488 diluted 1/1000
• Vectashield mounting medium

For NF186:

• Blocking solution: 5% Goat serum in PBS.
• Primary antibody: AF3235 Diluted 1/1000
• Secondary antibody: GAC488 diluted 1/1000
• Vectashield mounting medium

For CASPR1:

• Blocking solution: 5% Goat Serum in PBS
• Primary antibody: 133634 diluted 1/250
• Secondary antibody: GAR488 diluted 1/1000
• Vectashield mounting medium

For CASPR2:

• Blocking solution: 5% Goat Serum in PBS
• Primary antibody: Rabbit CASPR2 diluted 1/500
• Secondary antibody: GAR488 diluted 1/1000
• Vectashield mounting medium

Results: The samples were well transfected.