Perfuse mice with 4% PFA

Dissect out brains and post-fix in 4% PFA for 24 hours at 4⁰C

Wash brains in PBS 3 X 5 minutes (brains can be stored in PBS at 4⁰C until required)

Embed brains in 2% LMP agarose in H2O

Using a vibratome, glue brain in agarose down and cut 100 µm coronal sections of the desired brain regions

Carefully collect sections with a paint brush and move to wells of 24 well plate containing PBS

Transfer sections with brush to Eppendorf tubes contacting 1 mL antigen retrieval buffer

Transfer tubes to a heat block at 95⁰C for 20 minutes

Transfer sections from tubes back to PBS in the 24 well plate

Carefully using a pastette or P100 pipette remove PBS and replace 3x5 min PBS wash

Remove PBS and add block buffer for 2 hours at room temperature on an orbital shaker

Remove block and add primary antibodies in block solution for 24 hours at 4⁰C on a shaker

Remove primary antibodies and wash sections with PBS 3x1 hour at room temperature on shaker

Remove PBS and add species-specific Alexa fluorophore-conjugated secondary antibodies in block solution overnight 4⁰C on a shaker

Remove secondary antibodies and wash sections in PBS for 3x1 hour at room temperature

Stain with a nuclear marker for 20 minutes at room temperature

Mount sections on to superfrost slides using fluoromount G and secure with cover glass.

Image individual CNPase positive oligodendrocytes in the sparsely myelinated upper layers of the mouse cortex (Layers II-III) using a z step size of 0.5 µm. Only analyse cells with all complete myelin sheaths present within the 100 µm section as assessed by following each process from the cell body and ensuring none exited the section.

Trace and measure the length of the myelin sheaths in 3-dimernsions using ImageJ plugin – simple neurite tracer (Longair et al., 2011). Move through the z-stack and follow thin processes from the cell body to myelin sheaths – signal intensity will be much brighter in myelin sheaths compared to processes. Ends of sheaths can be defined where signal ends, can also be confirmed with caspr staining.

Solutions:

Antigen retrieval buffer (10mM citrate, 0.05% Tween):

Tri-sodium citrate 2.94 g in 0.95 Litre H2O, pH 6 then add 500 uL Tween 20 and top up to 1 L with H2O.

Block buffer (10% goat serum, 0.25% triton, in PBS)

Tested primary antibodies:

Image cells in the sparsely myelinated layer II-III

Thin CNPase positive processes connect cell body (asterisk) to myelin sheaths (arrowheads).