cFos Immuno
This protocol is for staining cFos as an index of neuronal activity.
This protocol was validated on tissue collected from C57BL6J mice 60-90 minutes after behavior. Tissue was extracted and directly placed in ice cold 4% pfa (i.e., no perfusion), stored at 4C overnight, and the next day transferred to 30% sucrose in 1x PBS. Once tissue sank, it was frozen at -80 prior to sectioning on a cryostat in 50 micron sections.
Primary antibody: Rabbit Anti-cFos (Synaptic Systems, 226 003)
Secondary antibody: Goat Anti-Rabbit AlexaFluor 488/568 (ThermoFisher, A11078/A11011)
Blocking solution: 3% normal goat serum, 0.3% Triton X-100, in PBS. For serum, the less expensive non-sterile stuff is fine
Note that all steps assume gentle agitation on shaker
- Incubate in blocking solution for 1 hour at room temp.
- Incubate overnight in primary antibody (1:2000; 1:5000 also works) diluted in blocking solution at room temp.
- Rinse 2x in PBS, 10 min each
- Incubate for 2-4 hours in secondary antibody (1:500) diluted in blocking solution at room temp.
- Rinse 3x in PBS, 10 min each
- Mount, coverslip, and enjoy
Comparison of home-cage controls vs animals exposed to novel environment.
