PROTOCOL: Immunohistochemistry in teased nerve fibers
Fixation: 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 triton 0.1% diluted in Goat Serum 5% (blocking solution). Incubate 1 hour at RT in a humid chamber.
Serum 1/100 in blocking solution –> incubate 1 h at TA in the humid chamber.
Commercial primary antibody anti-NF (AF3235 R&D System) 1/500 in blocking solution –> incubate 1 h at TA in the humid chamber.
Wash 2 times with PBS (5 min) with agitation
Secondary antibodies: GAC488 and GAH 594 1/1000 in blocking solution –> incubate 1 h at TA in the humid chamber.
