2018.05.28: Coating and seeding
- Prepare 2 plates of 60mm of diameter with 14 coverslips (12 mm)
- Treatment with Poly-D-Lysine (diluted 1/40 with PBS)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS 1x
- Treatment with Laminin (diluted 2 microL/ml PBS)
- Wash with PBS 1x
- Seed 15000 cells/cm2 –> every plate has 21 cm2 –> 315000 cells/plate 60 mm (in proliferation medium)
- Incubate at 37ºC for 24h
2018.05.30: treatment and medium exchange
Change the medium of the cells –> Add retinoic acid to the differentiation medium
*Change the half of the differentiation medium (+ RA) every 2 or 3 days.
ICC protocol:
- Try different fixation methods:
- Methanol -20ºC : 5 minutes
- PFA 4% : 10 minutes
- PFA 4%: 20 minutes
- Wash with PBS 1x
- Blocking solution: Goat Serum 5 % in PBS for 1h at RT.
- Primary antibodies (for each fixation):
- Anti-neurofilament (04-1112, Millipore). Diluted 1/500
- Positive control Serum (15-195) diluted 1/100
- Negative control Serum diluted 1/100
- Wash with PBS 1x
- Secondary antibodies:
- For anti-neurofilament: GAR488 IgG diluted 1/1000
- For serum: GAH594 IgG diluted 1/1000
- Wash with PBS 1x
- Vectashield mounting medium
Resultados:
- Fijando con metanol el Neurofilamento se ve mejor porque permeabiliza, y el neurofilamento está en el interior.
- Igualmente las células en todos los tipos de fijación se ven despegadas. Hay que probar diferentes métodos: hacer la immuno en placa de 24 wells, probar otros coatings (laminina sóla, Poly-L-lisina…), crécer células en chamber slides…
Se han hecho fotografías de las células después de la fijación (antes de hacer la ICC)
