Samples:
- Positive control (NF155+) 15-226
- Negative control
Both samples are tested in pre-fixed tissue (with PFA 4 %)
PROTOCOL:
- Defrost the slides
- 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 0.3% . Incubate 1 hour at RT in a humid chamber .
- Dilute the serum samples 1/100 in 5% goat –> add diluted serum and incubate 1 h at TA in the humid chamber.
- Dilute commercial primary antibody Chicken anti-human / rat / mouse-NF (AF3235 R & D System) * 1/500 with goat serum 5% –> Add the commercial diluted antibody and incubate 1 h at TA in the humid chamber.
- Wash 2 times with PBS (5 min) with agitation
- Secondary antibodies: dilute GAC488 and GAH 594 1/1000 with 5% goat serum –> incubate 1 h at TA in the humid chamber.
- Wash 2 times with PBS (5 min) with agitation
- Mount with Fluoromount
Result:
We can’t see the paranodes. Conclusion: we have to do the teasing without pre-fixation with PFA 4 %.
