This article describes a protocol for differentiating oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes and visualizing them using fluorescence imaging. The process involves the use of specific antibodies to identify cellular markers associated with oligodendrocyte maturation.
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 microliters of DPBS containing 5% NGS and 0.1% Triton X-100. Incubate the plate at room temperature for one hour with gentle orbital shaking. Prepare the primary incubation solution by diluting mouse monoclonal anti-MBP antibody and rabbit polyclonal anti-OLIG2 antibody in DPBS containing 5% NGS. Then, incubate the primary antibodies at 4 degrees Celsius overnight for 16 to 18 hours with gentle orbital shaking.
While washing the plate, prepare the secondary incubation solution by diluting the anti-mouse 680 and anti-rabbit 800 antibodies in DPBS containing 5% NGS. Next, aspirate the final wash and add 250 microliters of secondary incubation solution. Protect the plate from light, and incubate it at room temperature for one hour with gentle orbital shaking.
After that, scan the plate using an imaging system capable of detecting 700-nanometer and 800-nanometer fluorescence emissions. As a starting point, set the focal offset to 3 and sensitivities to 1.5 for MBP, 5.0 for actin, and 3.5 for OLIG2. Adjust the sensitivity values as needed to avoid signal overexposure.
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