20210218: Immunohisto-chemistry in teased nerve fibers in Amyotrophic neuralgia patients

Objective: To test IgG Amyotrophic neuralgia patients from Switzerland.

Samples: 21-2-0101, 21-2-0102, 21-2-0103, 21-2-0105, 21-2-0106, 21-3-0107.

PROCEDURE:

• Fixation: in acetone -20ºC  for 10 min at RT.
• Wash 2 times with PBS (5 min) with agitation.
• Use the marker (Pappen) 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.
• Wash 2 times with PBSx1 (5 min) with agitation.
• Serum 1/100 in blocking solution à incubate 1 h at TA in the humid chamber.
• Wash 2 times with PBSx1 (5 min) with agitation.
• Commercial primary antibody anti-NF (AF3235 R&D System)  1/500 in blocking solution à incubate  1 h at TA in the humid chamber.
• Wash 2 times with PBSx1 (5 min) with agitation.
• Secondary antibodies: GAC488 and GAH 594 1/1000 in blocking solution à incubate 1 h at TA in the humid chamber.
• Wash 2 times with PBS (5 min) with agitation
• Mount the preparation with Fluoromount.

RESULTS: All samples were positive for targetting of paranodes and nodes except the sample 21-2-0105,whose targetting was only positive for nodes.

Next to the image is indicated the magnification of the microscope lens (20x or 40x).