Samples: N33 (control) and N36 (case)
Immunohistochemistry (in brain frozen sections): 3 different IH
- IgG1 / IgG4
- Fix with acetone (5 min)
- Wash with PBS 1x (3 x 5 min)
- Block with 10 ml PBS + 0’2 g BSA) (30 min)
- Secondary antibodies (1 h):
- MAH biot. IgG1 diluted1/200
- MAH biot. IgG4 diluted1/200
- Wash with PBS 1x (3 x 5 min)
- Streptavidine 594 diluted 1/200
- Wash with PBS 1x (3 x 5 min)
- Fluoromount mounting medium
- NF – antiHuman
- Fix with acetone (5 min)
- Wash with PBS 1x (3 x 5 min)
- Block with Blocking Goat (10 ml PBS + 0’5 ml Goat Serum + 0’2 g BSA) (30 min)
- Primary antibody: anti-Neurofascin (1 h). Diluted 1/50 in blocking goat
- Wash with PBS 1x (3 x 5 min)
- Secondary antibodies (1 h): GAC 488 + GAH594 diluted 1/200 in blocking goat
- Wash with PBS 1x (3 x 5 min)
- Fluoromount mounting medium
- Calbindin – antiHuman
- Fix with acetone (5 min)
- Wash with PBS 1x (3 x 5 min)
- Block with Blocking Goat (10 ml PBS + 0’5 ml Goat Serum + 0’2 g BSA) (30 min)
- Primary antibody: anti-calbindin + alexa fluor 488 (1 h). Diluted 1/100 in blocking goat
- Wash with PBS 1x (3 x 5 min)
- Secondary antibody (1 h): GAH594 diluted 1/200 in blocking goat
- Wash with PBS 1x (3 x 5 min)
- Fluoromount mounting medium
Results:
- la tinció és inespecífica, perquè el cervell té biotina i la Streptavidina la reconeix.
- GAH594 fa tinció inespecífica i no es veu el mateix marcatge que amb GAH488
- l’Ac anti-calbindin + alexa fluor 488 no marca res. L’anticòs GAH594 també marca inespecíficament.
