2019.04.25: Seed cells (in proliferation medium)
- Coating with laminin 2’5 µg/ml diluted in Borate Buffer (for 1 h at 37ºC)
- Wash 2-3 times with PBS
- Seed aprox 10.000 cels/cm2 : 60 mm plate = 21 cm2
- In this case –> 6 plates of 60 mm with 200.000 cells (with 26 coverslips)
2019.04.26: Differentiation
- Change all the medium of the cells (differentiation medium)
- Each time the new differentiation medium is changed, 10 μM retinoic acid (RA) is added –> stock solution -80ºC (diluted in ethanol 96 %): 0,01 M –> put 1 µl in every ml of differentiation medium (if we change the half of the medium, we have to add the double of RA)
- Once the differentiation has begun, change the half of the differentiation medium every 2 or 3 days (for 6 to 8 days).
2019.05.02: ICC neuroblastoma neurons (day 7 of differentiation)
Samples: 101-148, negative control, positive control (15-198)
- Fix for 15 minutes with PFA 4 %
- Wash 3 times with PBS1x
- Block for 1 hour with Goat Serum 5 %
- Patient’s sera diluted 1/100 (1 h) –> in the box with parafilm, with the cells above
- Wash 3 times with PBS1x
- Secondary antibody: GAH488 IgG diluted 1/1000 (1 h) –> in the box with parafilm, with the cells above
- Wash 3 times with PBS1x
- Vectashield mounting medium
RESULT: Sample 114 and 124 were positive.
