Coating and seeding: 28.05.18 [see the post 20180601: ICC HTB-11 neurons (different cell fixation methods) ]
ICC protocol: 2 different protocols
- Fixation before the ICC
- Take one coverslip and put it in a 24 well plate
- Fix with PFA 4% (aprox 1 ml/well). Incubation at RT during 15 min.
- Wash with PBS 1x
- Primary antibody: anti-CNTN1. Diluted 1/1000 in Rabbit Serum 1/40
- Wash with PBS 1x
- Secondary antibody: RAG488 IgG diluted 1/1000 in Rabbit Serum 1/40
- Wash with PBS 1x
- Vectashield mounting medium
- Fixation after the Primary antibody incubation
- Take one coverslip and put it in a 24 well plate
- Primary antibody: anti-CNTN1. Diluted 1/1000 in differentiation medium (HTB11)
- Fix with PFA 4% (aprox 1 ml/well). Incubation at RT during 15 min.
- Wash with PBS 1x
- Secondary antibody: RAG488 IgG diluted 1/1000 in Rabbit Serum 1/40
- Wash with PBS 1x
- Vectashield mounting medium
Results: les cèl·lules es veuen més enganxades, tot i que encara es perden moltes durant les incubacions. El resultat és millor en el protocol 2 (fixar després d’incubar in vivo amb l’anticòs primari).
