Objective: To test sample 18-532 (positive in ICC for IgM towards NF140 and NF186) in IHC towards Neurofascin.
SAMPLES:
- 1 – 18-532
- 2.- CTRL –
PROTOCOL:
- Prepare a humid chamber with dampened sponges in an opaque box.
- Prepare a Hellendhal staining bucket full of acetone (-20 ° C).
- Remove the swine sciatic nerve teasing of the freezer and quickly put it directly into acetone.
- Wash with PBS 1x approximately 5 min with agitation (about 35 rev / min).
- Repeat the washing step.
- Dry the sample with a tissue without touching the actual sample.
- Use the marker to highlight the location of the sample without exerting excessive pressure.
- Cover the entire sample with Goat Serum 5% blockade solution with a plastic Pasteur pipette. Incubate 1 hour at RT in a humid chamber .
- Wash with PBS 1x approximately 5 min with agitation (about 35 rev / min).
- Dilute the serum samples 1/10 in 5% goat serum. Add 200 mcL /sample of diluted serum and incubate 1 h at TA in the humid chamber.
- Wash with PBS 1x approximately 5 min with agitation (about 35 rev / min).
- 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 with PBS 1x approximately 5 min with agitation (about 35 rev / min).
- Prepare the mixture of secondary antibodies. Dilute GAC488 antibody (green) 1/2000 and GAH- IgM-594 (red) 1/1000 with 5% goat serum in the same microtube. Add 500 mcL/ sample of the mixture of secondary antibodies and incubate 1 h in a humid chamber at TA.
- Wash approximately 5 min with agitation (about 35 rev / min). Important: avoid contact with light.
- Repeat the washing twice, covering the sample.
- Mount the preparation with Fluoprep: add two Fluoprep drops per sample and sealed them with a coverslip. Press to remove the excess and to ensure the homogeneity of the preparation. Carefully clean the edges and store protected from light.
RESULTS: The sample 18-532 -positive in ICC for IgM towards NF140 and NF186- have a positive result in the IHC but it marked the mielin, not the node. We are going to review if the patient had antiMAG antibodies and we are going to perform and IHC in peripheral nerve monkey.
