Apollo Half Time Oven 2X User Manual download pdf (Page 4)

Secondary antibodies for immunohistochemistry were from Vector Laboratories (horse anti-mouse

HRP-conjugated, 1 : 200, Cat# BA-2000 and horse anti-rabbit biotinylated; 1 : 200). Avidin-biotin-HRP

(ABC reagent) was prepared 30 min before use (2 drops of A + B per 5 ml PBS, Vector Laboratories)

and incubated for 30 min. PBS wash (3 x 5 min) was followed by DAB chromogen (1 drop of each

A+B+C + 1 ml H2O; DAB plus substrate kit, Invitrogen Cat#062020), water wash (2 – 5 min),

Counterstaining with Harris haematoxylin (20 seconds), wash in running tap water (2 – 5 min),

dehydratation in graded alcohol series (50%, 70%, 95%, 100%; 2 min each), xylene wash and

mounting in Permount (Fischer Cat#SP15-500).

Samples were incubated with the following secondary antibodies for immunofluorescence: goat

anti-mouse ALEXA 488-labeled (1 : 200, Cat#A11029, Invitrogen) and goat anti-rabbit ALEXA 594-

labeled (diluted 1 : 200, Cat#A11012, Invitrogen), and counterstained with HOECHST33342

(Invitrogen;for working solution dilution 1:1000 in PBS was prepared, 10 min), PBS washed (3x 5

min), mounted in Cytoseal60 and imaged immediately.

Primary antibodies were the same in both instances. SM Actin antibody was a mouse monoclone to

human SMA (working dilution 1: 100, clone 1A4, Dako Cat# M0851). CD68 antibody was a mouse

monoclonal to human protein (working dilution 1: 200; KP1; Santa Cruz Biotechnology Cat# sc-

20060). RTL1 was a rabbit polyclonal (working concentration 1.100) from Abcam (Cat# ab107990).

Statistical and bioinformatical analysis

NGS reads were mapped to human chromosomes to predefined genetic compartments (promoters

at-2000+500 from transcription start sites (TSS), exons and introns according to the genome update

GRCh37 coordinates provided by the ENSEMBL Genome Browser

(http://www.ensembl.org/index.html), and the number of unique reads per sample was

approximately 6 - 14 million out of 30 – 35 million raw reads. The counts for uniquely mapped reads

were normalized to represent a total of 10 million uniquely mapped reads per sample by adjusting

the number of reads at each compartment in the genome to the correct fractional amount given the

total reads mapped. After read count normalization statistically significant differences at specific loci

were identified. Fold-change differences in DNA methylation were defined for each genomic

compartment (atherosclerotic samples vs. normal) using locally installed HOMER software

(http://homer.salk.edu/homer/) at estimated false discovery rate FDR <0.05. Ingenuity Pathway

Analysis package (Ingenuity, Redwood City, California, USA) was used for bioinformatical analyses of

genes showing significantly (p<0.05) and prominently (cut off value was set to 2.5x fold difference)

modified DNA methylation patterns.

Microarray analysis (RMA normalization followed by filtering to remove weak signals and definition

of differential expression) was performed using Chipster program package.

The rest of the statistical analyses were performed using SPSS14 for Windows (SPSS Inc., Chicago,

Illinois, USA) and GraphPad Prism (GraphPad, La Jolla, CA, USA). Independent-samples T-test was

used for parametric variables and Mann-Whitney U-test for non-parametric variables. Correlations

between parametric variables were determined by Pearson’s algorithm. Categorical patient data was

analysed using Fisher Exact Test.

1 2 3 4 5

No comments

Apollo Half Time Oven 2X User Manual Page 4