Some immunostaining results selleck chemicals Palbociclib were analyzed as integrated density/intensity and normalized to values from controls of each experiment. Confocal microscopy (��40) was used for analysis of axonal degeneration (pNF-H and ��-APP immunofluorescence). For evaluation of apoptosis, TUNEL assay (Millipore) was performed according to the manufacturer’s instructions. Electron microscopy (EM) Animals were perfused with 4% PFA and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer. Coronal slices (1 mm thick) through the corpus callosum were cut in midsagittal plane and embedded in epon, oriented to visualize the entire cross-section of the midsagittal corpus callosum. Septostriatal sections were stained with uranyl acetate and lead acetate, and photographed as described previously (37).

The number of myelinated axons in multiple nonoverlapping fields (5 areas/section) for 3 mice in each group was counted with results expressed as a percentage of myelinated axons (number of myelinated axons/total axons �� 100). The g ratio was determined by calculating the ratio of the inner axonal diameter to the total outer diameter (inner axonal diameter plus surrounding myelin). At least 300 fibers were analyzed. Real-time RT-PCR studies Total RNA was isolated from the corpus callosum using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The purity and concentrations were determined using Gene Spec III (MiraiBio, South San Francisco, CA, USA). Reverse transcription was performed from 1 ��g total RNA in 20 ��l of diethylpyrocarbonate-treated water.

The complementary DNA (cDNA) at 1:10 dilution was used for SYBR Green real-time RT-PCR (Applied Biosystems, Foster City, CA, USA). Primers designed using the Primer3 software (http://frodo.wi.mit.edu/primer3/) were used for amplification for transcripts of MBP, proteolipid protein (PLP), platelet-derived growth factor (PDGFA), insulin-like growth factor-1 (IGF-1), tumor necrosis factor �� (TNF-��), interleukin-1�� (IL-1��), CCL2 (MCP-1), CCL5 (RANTES), S1P1, S1P5, and GAPDH (Supplemental Table S1). The expression of each gene was normalized by the corresponding amount of GAPDH mRNA for each condition. Relative amounts of each product were expressed as (2?��CT) relative to GAPDH or as 2?����CT relative to controls, as described in the ABI Prism 7300 Sequence Detection System user manual (Applied Biosystems).

At least 3 independent experiments were done, with each test condition in triplicates. Anacetrapib Western blot analysis Corpus callosum was homogenized in a lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% TritonX, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF, and 2 ��g/ml leupeptin. Samples (30 ��g/lane) were resolved by 12% SDS-PAGE and electroblotted to 0.45-��m PVDF membranes. After blocking with 5% nonfat milk in 0.