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Formaldehyde_fixed spinal cords were immersed in 30% sucrose until they sank.  Forty micron sections were cut on a freezing microtome and mounted on subbed slides.  Slides were dried in an oven at 50C for at least 2 hours.  The initial xylene step in published procedures for the eriochrome staining method was found to be unnecessary.  Therefore, thoroughly dried sections were hydrated in 95%, and 70% ETOH followed by dH2O (10 minutes each).    Sections were stained for 15 minutes using an eriochrome cyanine solution consisting of 2 ml 10% FeCl3 and 40 ml 0.2% eriochrome cyanine (sigma) in 0.5% aqueous H2SO4, brought to a final volume of 50 ml with dH2O (formula was increased six_fold for this tissue).

The crucial step in this procedure is differentiation.  Sections were differentiated by alternating between exposure to 0.1% NH4OH for 3_7 seconds and rinsing in dH2O for ~30 seconds until the blue background was reduced and cells turned faintly pink but still had blue shading.  Total time of exposure to NH4OH was approximately 20 seconds.

After the last rinse in dH2O, sections were dehydrated in 70%, 95%, and 100% EtOH (two changes), and three changes of xylene for 10 minutes each.  Coverslips were mounted using Krystalon.

The sections were photographed at 4x using a Nikon Coolpix 990 camera with an exposure of ½ second and F3.9.  Two procedures improved the contrast between the blue of axons and the red of somas.  First, we photographed sections at a low light level (low filament temperature probably resulted in an image with more red).  Second, blue_to_red contrast was improved in a single step using the "Auto Level" image adjustment in Photoshop.  A modest increase in color saturation further improved the images.

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