Each fraction was loaded as a single lane on separate12% polyacrylamide gels and transblotted onto Immobilon P. Membranes were stained with Ponceau S, cut into strips, and probed with various antibodies. Rabbit polyclonal antibodies for HDACs 1 5 and anti acetyl histone H4 ChIP grade rabbit antiserum were used at a 1 100 dilution. Goat anti rabbit secondary www.selleckchem.com/products/Axitinib.html anti bodies, conjugated to alkaline phosphatase, were used at a 1 500 dilution. Blots were color developed using NBT and BCIP and digitally scanned. Evaluation of transcripts in the retina by quantitative PCR Total retinal RNA was isolated at 1, 3, and 7 days post ONC and 2 ug was used for cDNA synthesis with reverse transcriptase and oligo. The resulting cDNA was diluted 10 fold and 1 ul was used for each qPCR reaction with SYBR Green PCR master mix and the appropriate HDAC primers, as described previously.

Each primer set was optimized, and the resulting amplimer cloned and sequenced to confirm identity. Quantitative PCR was conducted on triplicate samples in each run using an ABI 7300 Real Time PCR system. Tran script quantification was based on standard curves of each target amplimer and the absolute value for copy number was normalized to S16 ribosomal protein mRNA. The values are expressed as the ratio of crush control and normalized to the day 0 ratio that was set at 1. In addition to evaluating individual transcripts in sepa rate experiments, a qPCR mini array was developed that allowed us to examine 15 different mRNAs simultane ously during a single run.

Each 96 well plate of the array contained triplicate assays for 5 RGC specific genes, 4 genes that are abun dantly expressed in RGCs, and reportedly decline in expression, but are not necessarily RGC specific, 4 genes whose expression likely increase in damaged retinas, and 2 control genes. Two complete sam ples could be simultaneously examined on each plate. Primer design and PCR conditions were first normalized so that efficient amplification was obtained for all targets under the same PCR conditions. Sample quantification was carried out as described above, using a common standard curve for the entire plate. Immunofluorescence and quantification of AcH4 Indirect immunofluorescence on 5 um thick retinal cryo sections was done as described previously. Primary antibodies included H2AX, HDAC2, HDAC3, and AcH4 and were used at a 1 100 dilution.

Secondary antibodies used were Cilengitide goat anti rabbit with a Texas Red label and goat anti mouse with a FITC label. The images were obtained using a Zeiss Axioplan 2 Imaging microscope with Axiovision 4. 6. 3. 0 software and viewed in Adobe Photoshop. To quantify AcH4 staining, nuclear pixel intensity was measured in the ganglion cell and inner nuclear layers using the outline function of the Zeiss Axio vision software.