OBJECTIVE
Test patients that needed to be repeated from last experiment and check reactivity over Schwann cells fixed with PFA of those patients testing positive in the previous experiments. We also test several monoclonal antibodies to see if they are suitable to do double stainings.
PROCEDURE
We perform live staining in those patients that were not done or needed to be repeated and staining after fixation in patients that were already classified as positive. Monoclonal antibodies are tested with both live and fixed cells.
Live staining:
- Culture Schwann cells following protocol (Poly-D-lysine coated plates w/ coverslips up to 80% confluence in 60mm plates
- Transfer coverslips to 24-well plates (1 coverslip per well)
- Dilute CIDP serum (1/40) in Schwann cell medium and add to the wells
- We test these patients with live staining: JMEG, LGD, GDP, CTRL4, JVMG, MSH, RVT. Also test these monoclonal antibodies: anti-MPZ, anti-PMP2, antiCNTN1, antiCASPR1 and antiNeurofascin.
- Incubate 2h at 37ºC in incubator.
- Wash 3xPBS
- Fix with PFA 4% in PBS 10 minutes
- Wash 3xPBS
- Prepare secondary ( GAH IgG Alexa 488 1/500, DAR IgG Alexa 488 1/500 for PMP2 and MPZ, GAM IgG Alexa 488 1/500 for CNTN1 and CASPR1 and GAchicken IgY Alexa 488 1/500) and add to the coverslips
- Incubate 1h at RT
- Wash 3xH2O
- Mount with Vectashield with DAPI
PFA 4%-fixed cells staining
- Cells were harvested the day before, fixed 10′ with PFA 4%, washed 3xPBS, blocked 1 hour with Goat serum 5% in PBS and frozen.
- Thawed 2 60mm plates with 12 coverslips each and add 5mL of NGS 5% in PBS and let sit.
- Prepare primary antibodies:
- Patients: GDP, CTRL4, DLR, MIGP, MRVM, PVC, SSP, PPI, ATR, JRSO, MRE, MCS, TJPM, TMF. All at 1/40 in blocking.
- Monoclonal antibodies: anti-MPZ, anti-PMP2, antiCNTN1, antiCASPR1 and antiNeurofascin, all of them at 1/100 in blocking.
- Let 2h at RT
- Wash 3xPBS
- Add secondary Abs (Same as in live staining) and let 1h at RT away from light
- Wash 3xH2O
- Mount with Vectashield
RESULTS
The unspecific puctate staining in the cytoplasm that is presente in all patients and controls is only present in live staining. I believe that the union of imunoglobulins to Fc receptors and their internalization can be responsible for that pattern.
Two patients (JMEG and JVMG) have weak positivity. All others are negative.
None of the patients that were positive in live staining are positive in fixed cells at all (!!!!)
The antiCASPR and antiCNTN antibodies stain unspecifically the Schwann cell but only when fixed (!!!!). See images.
AntiCASPR1
ANTICNTN1
GDP
CTRL4
CONCLUSIONS
Fixed cells are not suitable for screening. GDP immunoprecipitation is ongoing.
We have to buy an antibody specific for Schwann cells.
