OBJECTIVE

Set up Schwann cell ICC to test the presence of antibodies against Schwann cells in CIDP patients

MATERIALS

• Schwann cell line (P3), Innoprot
• PFA 4% in PBS
• CIDP sera
• Secondary antibody (GAH Alexa 488)
• Vectashield

PROCEDURE

We follow to approches to see which condition is best: live cell staining and dead cell staining.

Live staining:

•  Culture Schwann cells following protocol (Poly-D-lysine coated plates w/ coverslips up to 80% confluence in 60mm plates
• Transfer coverslips to 12-well plates (1 coverslip per well)
• Dilute CIDP serum (1/100) in NGS 5% in PBS and add to the wells
• We used these sera: CLG, COC, SSP, DVE, AR, LAC, JMF, FXM and 2 controls (HD)
• Incubate 1h at 37ºC in incubator.
• Wash 3xPBS
• Fix with PFA 4% in PBS 5 minutes
• Wash 3xPBS
• Prepare secondary ( GAH IgG Alexa 488 1/500) and add to the coverslips
• Incubate 1h at RT
• Wash 3xPBS
• Mount with Vectashield with DAPI

Dead cell staining

Same approach but fixation w/ PFA is the first step and then ICC proceeds at RT with same antibodies and concentrations.

RESULTS

It looks like SSP serum reacts against Schwann cells, but the staining is only clear with live cells. All other patients, but LAC, seem to have reactivity against Schwann cells but weaker.

Controls don’t stain, although one of them has a very weak staining pattern that could resemble that of the positive patients, so it might be unspecific.

I haven’t seen any patient that has a pattern of staining similar to what was published in Brain by Kwa and co-workers (see reference)

Interestingly, LAC, that has antibodies against the glial form of neurofascin (NF155) does not stain. The Schwann cells we’re using are non-myelinating, so probably that’s key to see antigens.

CONCLUSIONS

We will make more patients and controls but using the same exact protocol they use in the cited paper

NOTES

Experiment performed by Fina.