PROTOCOLO IGUAL QUE TEASING
- ixation: in acetone -20ºC for 10 min at RT
- Wash 2 times with PBS (5 min) with agitation
- Use the marker (DakoPen) to highlight the location of the sample.
Indicate the sample number and the date in pencil on the frosted glass and place the samples in the humid chamber. - Blocking: cover the entire sample with Goat Serum 5% with triton 0.1% . Incubate 1 hour at RT in a humid chamber .
- Dilute the serum samples 1/100 in Goat Serum 5% with triton 0.1% –> add diluted serum and incubate 1 h at TA in the humid chamber.
- Wash 2 times with PBS (5 min) with agitation
- Dilute commercial primary antibody Chicken anti-human / rat / mouse-NF (AF3235 R & D System) * 1/500 with 5% with triton 0.1% –> Add the commercial diluted antibody and incubate OVER NIGHT AT 4ºc
- Wash 2 times with PBS (5 min) with agitation
- Secondary antibodies: dilute GAC488 and GAH 594 1/1000 with 5% with triton 0.1% –> incubate 1 h at TA in the humid chamber.
- Wash 2 times with PBS (5 min) with agitation
- Mount with Fluoromount
AJUSTES DEL PROTOCOLO EN ESTE CASO: Probamos diferentes anticuerpos comerciales
- 1. Caspr 1 1:60 Rabbit
- 2. LGI4 invitrogen 1:60 Rabbit
- 3. S100 Ab52642 1:100 Rabbit
- 4. Plexin 1:100 Rabbit
- 5. ADAM 22 1:100 Rabbit
- 6. NF 200 1: 200 Rabbit
- 8. Suero CNTN1+
- 7. Suero Caspr1+ CIDP78
Secundarios 1:500 (GAR o GAH 488)
RESULTADOS (VER FOTOS DE MARCAJES). El anticuerpos LGI4 y el S100 no marcan la mielina. ¿Podría ser por el tipo de fijación? En el paper de LGi4 utilizan formalina 10% y parafina
