Objective: To investigate Monkey’s Peripheral nerve IHC staining paterns of CIDP patients with CNTN1/Caspr1 Ab, a colocalization with Caspr1 and CNTN1 commercial mAbs
Samples:
- negative control 1:20
- 19186 1:20
- Piffort 1:20
- Regine 1:20
- CNTN1+ (15198) 1:20
a) IHQ protolocol with Caspr1 commercial mAb:
- We used commercial slides from Inova (peripheral nerve)
- Incubate with blocking solution (goat serum 5% in PBS) 1h
- Incubate 50 microl of sample (non-absorbed) 1h
- Wash 3 times with PBS 1x.
- Incubate with Caspr1 commercial mAb (1:20)
- Secondary Ab GAH AF488 monkey absorbed (1/400), GAR 594 for 2. 1 hour at RT.
- Wash 3 times with PBS 1x.
- Mount with Fluoromount mounting medium.
b) IHQ protolocol with CNTN1 commercial mAb:
- We used commercial slides from Inova (peripheral nerve)
- Incubate with blocking solution (rabbit serum 1/40 in PBS) 1h
- Incubate 50 microl of sample (non-absorbed) 1h
- Wash 3 times with PBS 1x.
- Incubate with CNTN1 commercial mAb (1:50)
- Secondary Ab RAH AF594 AND RAG AF488 (1/500) for 1 hour at RT.
- Wash 3 times with PBS 1x.
- Mount with Fluoromount mounting medium.
Results:
a) IHQ protolocol with Caspr1 commercial mAb:
- negative control–> negative
- 19186 –> paranodes, but the nerve is broken
- Piffort –> paranodes, colocalization with Caspr1 (FOTO confocal 4-10-19)
- Regine –> paranodes, colocalization with Caspr1 (FOTO confocal 4-10-19)
- CNTN1+ (15198) –> paranodes and staining of patched areas that could correspond to unmyelinated fibers
b) IHQ protolocol with CNTN1 commercial mAb: we are going to repeat this protocol using different secondary antibodies to see CNTN1 commercial antibody in red
- negative control –> negative
- 19186 –> paranodes
- Piffort –> paranodes
- Regine –> paranodes
- CNTN1+ (15198) –> paranodes and staining of patched areas that could correspond to unmyelinated fibers, that colocalized with CNTN1 commercial antibody.
