The horseradish peroxidase (HRP)-conjugated Caspase inhibitor secondary antibodies were purchased from Jackson Immunologicals (USA). All tissue preparation was carried out essentially as described previously (Bhat et al., 2001, Pillai et al., 2009 and Thaxton et al., 2010). Briefly, SNs harvested from anesthetized mice were fixed in 4% paraformaldehyde (in phosphate buffered saline; PBS) for 15–30 min at 4°C, followed by extensive washing in PBS. The SNs were subsequently teased

onto slides and dried overnight. The teased SNs were subjected to permeabilization in ice-cold acetone for 15 min followed by washing in PBS, and were blocked in buffer (5% BSA, 1% NGS, and 0.2% Triton X-100, in PBS) for 1 hr. The SNs were incubated with primary antibodies overnight in blocking buffer, followed by rinsing in PBS

before RO4929097 datasheet being incubated with secondary antibodies for 1 hr at room temperature (RT). Finally, the SNs were mounted in VectaSheild (VectorLabs) before imaging. Spinal cord tissue was prepared as follows: mice were anesthetized and pericardially perfused with 4% paraformaldehyde (in PBS). The spinal cord was removed and either subjected to postfixation in 4% paraformaldehyde overnight at 4°C or washed in PBS. Following postfixation the spinal cords were washed in PBS and sliced using a Vibratome (Leica), to 30 μm in thickness. The spinal cord sections were blocked as described above, incubated with primary antibodies overnight at 4°C with shaking, washed, and subjected to secondary antibodies. All images were acquired under Protein kinase N1 identical settings using a Bio-Rad Radiance 2000 confocal microscope with Zeiss software as previously described (Thaxton et al., 2010), or using an Axiovert scanning confocal microscope using Zeiss LSM510 software. SNs and spinal cords from age-matched

wild-type (+/+), Nefl-Cre;NfascFlox, and Cnp-Cre;NfascFlox mice were harvested and frozen at −80°C until processing. The extraction was performed as follows: tissues were homogenized in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 10 mM EDTA [pH 8.0], 1% SDS, 1% Triton X-100, and a protease inhibitor cocktail tablet) by 25–50 stokes using a glass mortar and pestle. The homogenates were incubated for 45 min on ice with occasional mixing. The resulting lysates were centrifuged at 13,000 rpm for 20 min at 4°C, and the supernatant was collected. Protein concentration was determined by Lowry Assay (BC assay, BIORAD), and equal amounts of protein were resolved by SDS-PAGE, followed by transfer of the proteins onto nitrocellulose membranes. The membranes were then blocked (5% nonfat dry milk in PBS with 0.1% Tween-20 [PBS-T]), incubated with primary antibodies for 1 hr at RT or overnight at 4°C, and washed in PBS-T before secondary antibodies were applied for 40 min at RT. The membranes were washed further in PBS-T before chemiluminescent substrate was applied. The membranes were then exposed to autoradiographic film to obtain a signal.