Tissue sections were fixed in 10% buffered formalin, dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin. Five-micron tissue sections were subjected to Masson’s Trichrome staining at the Research Histopathology Core at the UTMB, and fibrosis was assessed by measuring the collagen area as a percentage of the total myocardial area (n = 4 mice /group, 2–3 slides per mouse, 10 microscopic fields per slide) using Simple PCI software (v.6.0; Compix, Sewickley PA). Sections were scored based on percent of fibrotic area: (0) <1%, (1) 1–5%, (2) 5–10%, (3) 10–15%, and (4) >15% .
Mix… and then don’t match. Un-match, mismatch, colour block, do anything but go matchy matchy. Just as Kareena Kapoor defended her two dissimilar pair of red heels in Kabhi Khushi Kabhi Gham. While being poked fun at by Hrithik Roshan she defended her fashion blunder with the trademark line, “That’s the latest trend.” Well, twenty years on, she couldn’t have been more apt.
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Tissue or cell lysates (1 mg/ml protein) in IP incubation buffer (Active Motif, Carlsbad CA), were incubated overnight at 4°C with 20 ?g of antibodies against POLG, PARP1, or PAR molecules. Samples were then loaded on to Protein G Agarose Prepacked Columns (Active Motif), and columns were washed five times with IP wash buffer (Active Motif), to remove non-binding proteins. Immune complexes were eluted from the columns in 0.2 M glycine (pH 2.5) solution. The eluents were pooled (total 100 ?l), neutralized with 20 ?l of 1 M Tris-HCl (pH 9.0), and used for Western blotting.
For ChIP assay, Imprint Chromatin Immunoprecipitation Kit was employed (Sigma). Briefly, Hela cells (5 X 107) or tissue sections (10 mg) were incubated with 9 ml of 1% buffered formaldehyde for 30 min at room temperature on a rocking platform to cross-link DNA/proteins, neutralized by adding 1 ml of 1.25 M glycine buffer, homogenized, and then sonicated on a Misonix XL2020 sonicator (30 pulses, 30 sec on/off, 100% power) to fragment the chromatin DNA. Cross-linked samples were subjected to immune-precipitation with mouse hPARP1 N-terminus-specific monoclonal antibody (sc-74470, Santa Cruz). Mouse IgG (Sigma) was used as negative control. The formaldehyde cross-link was reversed by incubating for 15 min at 65°C. Samples were then treated with RNase H and protease K, to digest RNA and proteins, respectively, and DNA was extracted with phenol/chloroform and purified by using QIAquick PCR Purification Kit (28104, Qiagen). ChIP DNA was used as substrate for PCR amplification of PARP1-bound sequences by using the oligonucleotides listed in S1 Table.