20191029: IHC IgG Swine sciatic nerve teasing in patients CNTN1/Caspr1+ (Oxford samples):

Samples:

1. negative control
2. positive control
3. 19200
4. 19204
5. 19209
6. 19212
7. 19216
8. Regine CNTN1 pre-absorbed
9. Regine caspr1 pre-absorbed
10. Regine CNTN1/Caspr1 pre-absorbed
11. 19203
12. 18806

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 0.1% . Incubate 1 hour at RT in a humid chamber .
• Dilute the serum samples  1/100 in Goat Serum 5% with triton 0.1% –> add diluted serum and incubate 1 h at TA in the humid chamber.
• Wash 2 times with PBS (5 min) with agitation
• Dilute commercial primary antibody Chicken anti-human / rat / mouse-NF (AF3235 R & D System) * 1/500 with 5% with triton 0.1% –> 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% with triton 0.1% –> incubate 1 h at TA in the humid chamber.
• Wash 2 times with PBS (5 min) with agitation
• Mount with Fluoromount

Results:

1. negative control –> NEGATIVO
2. positive control –> PARANODO
3. 19200 –> MARCAJE AXONAL INESPECÍFICO
4. 19204 –> PARANODOS ++
5. 19209 –> PARANODOS+
6. 19212 –> PARANODOS+
7. 19216 –> PARANODOS+
8. Regine CNTN1 pre-absorbed –> NEGATIVO
9. Regine caspr1 pre-absorbed –> NEGATIVO
10. Regine CNTN1/Caspr1 pre-absorbed –> NEGATIVO
11. 19203 –> NEGATIVO
12. 18806 –> NEGATIVO