Dfc480 digital camera

Nerves were freshly dissected, fixed in 2% glutaraldehyde in phosphate buffer, osmicated in 1% OsO₄, alcohol dehydrated, infiltrated with propylene oxide and embedded in Epon. Transverse semithin sections and ultrathin sections were cut with an Ultracut microtome [11 (link),78 (link)]. Semithin sections were stained with toluidine blue and acquired with a Leica DM5000 microscope equipped with a DFC480 digital camera, whereas ultrathin sections were stained with lead citrate and photographed with a Zeiss (Oberkochen, Germany) EM10 electron microscope. g-ratio (axon diameter/fiber diameter) was measured on semithin sections with semi-automated computer based morphometric analysis using Leica QWin V3 software [11 (link)]; four-six images per nerve were acquired with a 100x objective; ~800–2000 fibers per condition were measured. On EM images, g-ratio was measured using ImageJ software. 50–70 myelinated fibers from 10–12 images per animal were analyzed, from three mice per genotype. The number of demyelinated (naked) axons was counted blind to genotype on images acquired with a 100x objective from sciatic nerve semithin sections. The number of onion bulbs was measured on entire quadriceps nerves: single images were acquired with a 40x objective and nerves were reconstructed with Adobe-Photoshop CS4 (Adobe Systems, San Jose, CA). Three-five animals per genotype were used.