Dual Fluorescence Imaging to Identify Differentiated Oligodendrocytes

Take a culture of oligodendrocyte precursor cells (OPCs), isolated from a rat pup brain, that have been induced to differentiate into oligodendrocytes. The cells are fixed to preserve cellular architecture.

Differentiated oligodendrocytes express myelin basic protein, or MBP, a marker of mature oligodendrocytes, whereas both OPCs and differentiated cells express oligodendrocyte transcription factor 2, or Olig2.

Apply a permeabilization-blocking solution and incubate with agitation, permeabilizing cellular membranes and blocking non-specific binding sites.

Add primary antibodies targeting the markers and incubate with agitation to allow binding.

Wash to remove unbound antibodies, then add different fluorophore-conjugated secondary antibodies specific to the primary antibodies and incubate with agitation.

Wash the cells, then place them in a dual fluorescence imaging system.

Use light of specific wavelengths to excite the distinct fluorophores and detect the emitted signals to visualize the markers.

Identify differentiated oligodendrocytes expressing olig2 and MBP among the undifferentiated OPCs expressing only olig2.

Bring the plate to room temperature. Aspirate the wells, and add 250 mic