OBJECTIVE
To test reactivity against DRG neurons in patients with CIDP
MATERIALS
- DRG neurons plated on coverslips the on 12.06.2013. We followed Liu et al protocol (PLoS One. 2013;8(4):e60558) modified by Fina.
- CIDP sera from our collection
- Secondary Ab GAH IgG Alexa Fluor 488
- PFA 4% in PBS
- Vectashield
PROCEDURE
Did the experiment in two days
- DAY 1: COC, CPS, RMJ, DCG, ATR, JVMG, ACP, GDP, CTRL3, CTRL4
- DAY 2: CLG, AR, FLS, DVE, JMF, CAP, JPL, MPG, GMM, MSP, MBM, JCR, BTA, CCC, JTF, PSP, FXM, FBL, JMC, MJJ, DTS, SISR, GRG, UEJ, MML, MRVM, MLR, MCRA, DLR, TMF
- Washed old media and substitute with 200 uL of new media
- Add serum (4uL) to each well with a coverslip
- Let 1 hour incubation at 37ºC
- Remove medium and wash 3x with PBS
- Add 4% PFA in PBS and let 5 minutes.
- Wash 3xPBS
- Add secondary antibody GAH IgG 488 1/500 diluted in 5% NGS in PBS and incubate 1 hour
- Wash 3xPBS
- Mount with Vectashield with DAPI
RESULTS
Positive controls (patients with contactin-1 antibodies) show a nice staining that i would say it’s even better than with hippocampal neurons.
See positive results in database. We’ve found several positive patients that were not positive with hippocampal neurons. Some of the positives are clear candidates for IP (CAP, UEJ)
Image is example of positive patient (JMF). More images stored.
CONCLUSIONS
DRG neurons work for antigen discovery at least as well or even better than hippocampal neurons. We’ll start IPing JMF/UEJ
