OBJECTIVES: To confirm positive result on ICC.
SAMPLES: 23, 24, CTRL – and CTRL + (CNTN1: 15-198)
PROTOCOL:
- Fixation: in acetone 10 min at RT
- Wash 2 times with PBS (5 min) with agitation
- Use the marker (DakoPen) to highlight the location of the sample without exerting excessive pressure.
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% blockade solution with triton 0.3% . Incubate 1 hour at RT in a humid chamber .
- Dilute the serum samples 1/100 in 5% goat –> add 200 mcL /sample of 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 200 mcL /sample of 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 the preparation with Fluoromount
RESULTS: The samples were negative and the positive control was positive (we can see the paranodes). The primary antibody has a very mild staining; we don’t know if it is a problem with the primary antibody or with the GAC488. We will have to repeat the experiment to make some photographies for the article. NOTE: Repeat it with CASPR antibody and use the patient that is positive for nodal neurofascin as positive control.
