Anti desmin antibody

Four to eight hours after irradiation, the fixed specimens were cryoprotected in 30% sucrose solution for 24 h and frozen in 2-methylbutane. The horizontal sections (20–30 µm, CM3050S, Leica Biosystems Nussloch GmbH, Nußloch, Germany) were treated for 10 min at 95 °C with citrate buffer pH 6.0 (antigen retrieval), then washed three times and permeabilized with TritonX-100-Solution (Sigma Aldrich, St. Louis, MO, USA). After blocking with 10% normal-goat-serum (NGS, Sigma Aldrich, St. Louis, MO, USA) for 1 h, the specimens were treated with anti-gamma H2AX antibody (1:200; ab2893, Abcam, Cambridge, UK) or anti-desmin antibody (Sigma Aldrich, St. Louis, MO, USA) overnight at 4 °C. After several washes the samples were incubated for 2 h at 37 °C with the secondary goat anti-rabbit IgG Cy3 antibody (1:200; Invitrogen A10520). The slices were washed three times, covered with ProLong Gold antifade mountant with DAPI (Invitrogen, Waltham, MA, USA), and viewed and quantified with LAS software on a Leica DMI6000 microscope.

The findings in this report rely heavily on the double-staining of cultured cells with Rhodamine (Rho)- or fluorescein (FITC)-phalloidin (1:40 and 1:10, respectively; Molecular Probes) and one of the following antibodies: anti-sarcomeric-α-actinin (s-α-actinin) antibody (1:2500, clone EA-53, Sigma), anti-β-actin (1:800, clone AC-15, Sigma), anti-titin Z1Z2 antibody (1:30, [38 (link)]), anti-C-protein (1:50, clone MF-1) that stains the A-bands of striated myofibrils [39 (link)], anti-nebulin M177M181 antibody (1:50, [40 (link)]), anti-desmin antibody (1:200, Sigma), anti-tyrosylated-α-tubulin antibody (undiluted, clone YL1/2), anti-vinculin antibody (1:50, clone VIN-11-5, Sigma), anti-paxillin antibody (1:50, BD Transduction Labs.), anti-β1-integrin antibody (1:10, Sigma), anti-talin antibody (undiluted, clone 8e6, Iowa Hybridoma Bank), anti-cadherin 2 (N-cadherin) antibody (1:5, BioGenex), anti-Mrf4 (Myf6) antibody (1:100, Santa Cruz Biotechnology), anti-Arp2/3 antibody (1:100, Abcam), and anti-vimentin antibody (1:100, clone Vim 3B4, Boehringer Mannheim).