Computational protocol: Coordinate activities of BRD4 and CDK9 in the transcriptional elongation complex are required for TGFβ-induced Nox4 expression and myofibroblast transdifferentiation

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Protocol publication

[…] Tissue sections of skin biopsies from HTS and NBS regions were deparaffinized and rehydrated and antigen retrieval was performed with 10 mM sodium citrate, pH 6. Sections were blocked with 5% goat serum in TBS-T for 1 h and incubated with primary antibody overnight at 4 °C. After washing three times with TBS-T, sections were incubated with a fluorescent secondary antibody (AF-568 goat anti-rabbit) for detection of αSMA or a biotinylated secondary antibody for detecting TGFβ for 1 h. For detection via immunofluorescence, tissue sections were washed again with TBS-T, incubated with DAPI (Molecular Probes) for nuclear counterstaining and glass coverslips were mounted using Dako Fluorescence Mounting Medium (Carpenteria, CA, USA). For chromogen-based detection, tissue sections were incubated with avidin–biotin complex (Vector Labs, PK6101) followed by exposure to DAB substrate (Vector Labs, Burlingame, CA, USA, SK4100) and counterstaining with hematoxylin. Sections were dehydrated with serial washes in ethanol and xylene before mounting of coverslips. Immunostained sections were evaluated using a Nikon Eclipse 80i microscope (Melville, NY, USA) and images were captured with an attached Nikon DSM1200F digital camera. ImageJ (NIH, Bethesda, MD, USA) software was used to make composite images and to add the scale bar. [...] Experiments were repeated at least three times, and the mean±S.E.M. of n=3 experiments was plotted, unless otherwise noted. This sample size was selected based on effect sizes and experimental variability. Differences across multiple groups were analyzed by analysis of variance, followed by Newman–Keuls or Tukey's pairwise comparison. SigmaPlot 12.5 (Systat Software Inc., San Jose, CA, USA) was used for analyzing and graphing data. P<0.05 was considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Chemicals Oxygen