20191009: Monkey’s peripheral nerve IHC in CIDP patients

Objective: To investigate Monkey’s Peripheral nerve IHC staining paterns of CIDP patients with CNTN1/Caspr1 Ab, a colocalization with CNTN1 commercial mAb

Samples:

1.  negative control 1:20
2. Piffort 1:20
3. Regine 1:20
4. 16047 1:20
5. CNTN1+ (15198) 1:20

b) IHQ protolocol with CNTN1 commercial mAb:

• We used commercial slides from Inova (peripheral nerve)
• Incubate with universal blocking solution 1h
• Incubate with CNTN1 commercial mAb (1:50)
• Wash 3 times with PBS 1x.
• Secondary Ab DAG AF555 (1/500) (DAG IgG H+L A-21432 Invitrogen) 1h
• Wash 3 times with PBS 1x.
• Incubate with Goat serum 5% in PBS blocking solution 1h
• Incubate 50 microl of sample 1:20 (non-absorbed) 1h
• Wash 3 times with PBS 1x.
• Secondary Ab GAH Monkey Abs AF488 (1/400) for 1 hour at RT.
• Wash 3 times with PBS 1x.
• Mount with Fluoromount mounting medium.

Results:

1.  negative control 1:20 –> negative
2. Piffort 1:20 –> paranodes
3. Regine 1:20 –> paranodes
4. 16047 1:20 –> paranodes
5. CNTN1+ (15198) 1:20 –> paranodes and and staining of patched areas that could correspond to unmyelinated fibers, that colocalized with CNTN1 commercial antibody.  THIS PROTOCOL WORKED BETTER THAN THE PREVIOUS ONE. WE MADE PHOTOS WITH CONFOCAL MICROSCOPE.