20190228: IHC IgG Swine sciatic nerve teasing (16-134 and test with PFA4% prefixation)

Samples:

1. 16-134
2. Positive control (CNTN1+) 15-198
3. Negative control
4. Positive control (NF155+) 15-226
5. Negative control

1-3: done in fresh teased fibers (not fixed with PFA 4%); 4-5: done in prefixed teased fibers (with PFA 4%).

PROTOCOL:

• 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 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

Results: the IHCs done in not prefixed tissue are better than the ones done in prefixed tissue.

1. Positive
2. Positive
3. Negative
4. we can’t see the paranodes
5. we can’t see the paranodes