20180409 – 20180411: Coating and ICC in HTB-11 cell line (MS patients)

2018.04.09:

• Prepare 1 plate of 60mm of diameter with 24 coverslips (9 mm)
• Treatment with Poly-D-Lysine (diluted 1/40 with boratte buffer)
• Incubate for 1h (37ºC, 5% CO2).
• Wash with PBS 1x (4 times)
• Trypsin-EDTA a plate of HEK293.
• For every plate: 5ml of HEK medium + the volume that correspond to 1×10⁶ of cells
• Incubate at 37ºC for 48h.

2018.04.11:

• Fix with PFA 4% . Incubation at RT during 10min.
• Wash with PBS 1x
• Blocking solution: 5% goat serum in PBS for 1h at RT.
• Remove the blocking solution:
  • 14 coverslips: ICC
  • 10 coverslips: freeze at -80ºC

ICC:

Samples: 

• Patient’s sera diluted 1/100 with 5 % Goat Serum.
• Secondary antibody: GAH594 diluted 1/1000
• Vectashield mounting medium

Results: pacientes 102 y 103 positivo débil (sólo se ve marcage débil en algunas células). El resto de muestras negativas.