Were determined and processed.ImmunohistochemistryFrozen 10-mm-thick sections of isolated retina samples obtained from normotensive eyes and hypertensive eyes after IOP elevation were fixed in cold acetone for 10 min. They were washed three times for 5 min each in PBS and blocked with 10 fetal calf serum (FCS) for 30 min. The sections were then incubated overnight at 4uC with a primary antibody, polyclonal anti-rabbit b crystallin (gift from the Department of Biochemistry, Hyderabad, India), which was diluted at 1:400 in 10 FCS. After rinsing the slides three times each in PBS for 5 min, the sections were incubated with the secondary anti-rabbit Cy2 antibody (Dianova, Hamburg, Germany) diluted at 1:200 in 10 FCS for 30 min at room 18334597 temperature, and then washed three times for 5 min each in PBS. Finally, the slides were coverslipped with Mowiol (Hochst, ?Frankfurt, Germany). The nuclei of retinal cells were stained by adding 49,6-diamino-2-phenylindole dihydrochloride hydrate (Sigma-Aldrich) to the Mowiol embedding medium. Slides were examined with the aid of a fluorescence microscope (Axiophot, Carl Zeiss) with the appropriate filters. Negative controls comprised sections processed without addition of the primary antibodies. Control and experimental sections were stained simultaneously to avoid variations in immunohistochemical staining.from an in-house MWG Biotech expressed sequence tag 23388095 sequencing project. To design microarrays with optimal hybridization conditions, existing databases are filtered for redundant sequences and the oligonucleotides are designed with the Oligos-4-Array (developed by MWG Biotech). This requires that nontarget genes be less than 75 similar over a 50-base target region. In fact, if the 50-base target region is marginally similar (50?5 ), it must not include a stretch of complementary sequence of .15 contiguous bases. The oligonucleotide design thus guarantees the exclusion of both dimer and secondary structure formation. Cross-hybridization is minimized by exhaustive BLAST and global Smith-Waterman searches. The microarrays were scanned at a resolution of 10 mm at three photomultiplier gain settings in order to optimize the dynamic range. The resulting three images were integrated into one intensity value for each spot using the software packages ImaGene and GeneSight (MWG Biotech), and MAVI (MWG Biotech). The fluorescent signals were corrected and normalized for the difference between Cy3 and Cy5. Samples from each of the three cohybridizations were compared independently of each other. The signal values of probe sets that were reliably detected in most of the GW 0742 cost experiments in each group were used in two-sample, two-tailed ttests between the “experimental” and “control” groups (nonglaucomatous vs. glaucomatous retina). Probe sets were selected from candidate genes using a t-test based on p,0.05, and the ratio of means (relative change) between the two groups was Pentagastrin supplier calculated with “control” as the denominator. The final relative changes quoted here are the average values of three independent experiments. The cut-off values for up- and down-regulation were set at .3.0-fold and ,0.3-fold, respectively. The biological function of differentially expressed genes with a change of .3.0fold or ,0.3-fold were modeled according to their biological process using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification system (Applied Biosystems, San Diego, CA, USA). The PANTHER classificatio.Were determined and processed.ImmunohistochemistryFrozen 10-mm-thick sections of isolated retina samples obtained from normotensive eyes and hypertensive eyes after IOP elevation were fixed in cold acetone for 10 min. They were washed three times for 5 min each in PBS and blocked with 10 fetal calf serum (FCS) for 30 min. The sections were then incubated overnight at 4uC with a primary antibody, polyclonal anti-rabbit b crystallin (gift from the Department of Biochemistry, Hyderabad, India), which was diluted at 1:400 in 10 FCS. After rinsing the slides three times each in PBS for 5 min, the sections were incubated with the secondary anti-rabbit Cy2 antibody (Dianova, Hamburg, Germany) diluted at 1:200 in 10 FCS for 30 min at room 18334597 temperature, and then washed three times for 5 min each in PBS. Finally, the slides were coverslipped with Mowiol (Hochst, ?Frankfurt, Germany). The nuclei of retinal cells were stained by adding 49,6-diamino-2-phenylindole dihydrochloride hydrate (Sigma-Aldrich) to the Mowiol embedding medium. Slides were examined with the aid of a fluorescence microscope (Axiophot, Carl Zeiss) with the appropriate filters. Negative controls comprised sections processed without addition of the primary antibodies. Control and experimental sections were stained simultaneously to avoid variations in immunohistochemical staining.from an in-house MWG Biotech expressed sequence tag 23388095 sequencing project. To design microarrays with optimal hybridization conditions, existing databases are filtered for redundant sequences and the oligonucleotides are designed with the Oligos-4-Array (developed by MWG Biotech). This requires that nontarget genes be less than 75 similar over a 50-base target region. In fact, if the 50-base target region is marginally similar (50?5 ), it must not include a stretch of complementary sequence of .15 contiguous bases. The oligonucleotide design thus guarantees the exclusion of both dimer and secondary structure formation. Cross-hybridization is minimized by exhaustive BLAST and global Smith-Waterman searches. The microarrays were scanned at a resolution of 10 mm at three photomultiplier gain settings in order to optimize the dynamic range. The resulting three images were integrated into one intensity value for each spot using the software packages ImaGene and GeneSight (MWG Biotech), and MAVI (MWG Biotech). The fluorescent signals were corrected and normalized for the difference between Cy3 and Cy5. Samples from each of the three cohybridizations were compared independently of each other. The signal values of probe sets that were reliably detected in most of the experiments in each group were used in two-sample, two-tailed ttests between the “experimental” and “control” groups (nonglaucomatous vs. glaucomatous retina). Probe sets were selected from candidate genes using a t-test based on p,0.05, and the ratio of means (relative change) between the two groups was calculated with “control” as the denominator. The final relative changes quoted here are the average values of three independent experiments. The cut-off values for up- and down-regulation were set at .3.0-fold and ,0.3-fold, respectively. The biological function of differentially expressed genes with a change of .3.0fold or ,0.3-fold were modeled according to their biological process using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification system (Applied Biosystems, San Diego, CA, USA). The PANTHER classificatio.
