20180713: ICC HTB11 Neurons (CIDP and MS patients – IgG)

2018.07.02: Coating and seeding

• Prepare 4 plates of 60 mm of diameter with 14 coverslips (12 mm)
• Treatment with laminin  1h (2’4 microg/ml diluted in Borate Buffer)
• Wash with PBS 1x (3 times)
• Seed 30000 cells/cm2 –> every plate has 21 cm2 –> 630000 cells/plate 60 mm (in proliferation medium)
• Incubate at 37ºC

2018.07.06: treatment and medium exchange

Change the medium of the cells –> Add retinoic acid to the differentiation medium (1 microl RA in 1 ml differentiation medium)

*Change the half of the differentiation medium (+ RA) every 2 or 3 days.

2018.07.13: ICC

Samples: 

ICC Protocol:

• Take 48 coverslips of the plates and put them in 24 well plates
• Fix with PFA 4% . Incubation at RT during 15 min.
• Wash with PBS 1x
• Block with Goat Serum 5 % (incubation at RT during 1 h)
• Patient’s sera diluted 1/100  with Goat Serum 5 %
• Wash with PBS 1x
• Secondary antibody: GAH 488 IgG diluted 1/1000  in Goat Serum 5 %
• Wash with PBS 1x
• Vectashield mounting medium

Results: