AIM: to retest for anti-CNTN1 antibodies in patients with CIDP (CIDPRIT cohort) at baseline (for technical reasons the CNTN1 transfection of these samples was not correct).
MATERIALS REQUIRED:
- Cells (cultured on coverslips or chamber slides)
- Fixative (4% paraformaldehyde)
- Blocking buffer (Rabbit serum 1/40 for CNTN1).
- Primary antibody (anti-CNTN1).
- Secondary antibody conjugated to a fluorophore (RAG488 and RAH594 for CNTN1).
- Mounting medium (Fluoromont)
- PBS (phosphate-buffered saline) 1x
SAMPLES: 5 Samples from the Italian CIDP cohort at baseline, 1 positive control for CNTN1.
PREPARATION OF CELLS:
- Grow transfected cells with the protein of interest (CNTN1) on chamber slides.
FIXATION:
- Fix cells with the fixative solution (4% paraformaldehyde) for 10 minutes at room temperature.
- Rinse cells with PBS to remove fixative.
BLOCKING:
- Block nonspecific binding sites by incubating cells in blocking buffer for 60 minutes.
- Wash cells 3 times with PBS.
PRIMARY ANTIBODY INCUBATION:
- Dilute the primary antibody in blocking buffer (1/500).
- Incubate cells with the primary antibody solution for 1 hour at room temperature.
- Wash cells 3 times with PBS to remove unbound primary antibody.
SECONDARY ANTIBODY INCUBATION:
- Dilute the secondary antibody conjugated to a fluorophore in blocking buffer (1+1/500).
- Incubate cells with the secondary antibody solution for 1 hour at room temperature in the dark.
- Wash cells 3 times with PBS to remove unbound secondary antibody.
MOUNTING:
- Mount coverslips on glass slides using mounting medium.
IMAGING:
- Visualize cells using a fluorescence microscope equipped with appropriate filters for the fluorophores used.
RESULTS:
24-2-0232: negative
24-2-0235: negative
24-2-0252: negative
24-2-0255: negative
24-2-0258: negative
