What immunofluorescent labeling protocol do you recommend for iCell DopaNeurons?

We have an Application Protocol for immunofluorescent labeling of iCell® DopaNeurons, which has been used with a variety of neuronal proteins.

General Protocol Recommendations:

  • Assay ECM: Fresh PLO laminin double coat
  • Assay density: 64,000 cells/well in a 96-well plate (200,000 cells/cm2)
  • Assay media: Complete iDopaNeuron Maintenance Medium (needs both Medium Supplement and Nervous System Supplement to complete media)
  • Fixation buffer: 4% formaldehyde in PBS
  • Blocking buffer: 2% donkey serum, 2% fetal bovine serum, and 0.2% Triton X-100 in D-PBS
  • Washing buffer: 2% fetal bovine serum in D-PBS

Assay timing: On days 7, 14, and 21 post-thaw we stained cells positive for TH; on day 14 post-thaw we stained for other neuronal and midbrain specific markers (see ICC protocol); while vesicular protein staining will need to occur later, we cannot recommend an exact time point but suggest trying earliest at day 14 or later post-thaw, similar to neurons.

References:

  1. iCell DopaNeurons User's Guide
  2. Application Protocol: Immunofluorescent Labeling