20210216: Immunohisto-chemistry in teased nerve fibers in Amyotrophic neuralgia patients

Objective: To test IgG Amyotrophic neuralgia patient of Hospital de Sant Pau and a positive control of NF155.

PROCEDURE:

Samples: 370392 and NF155+.

• 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: The control positive of NF155 was positive for targetting of paranodes. However, the other sample was negative. The last result was unexpected.

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