Cytofix cytoperm solution

Cells were fixed and permeabilized with BD Cytofix/Cytoperm™ solution (BD biosciences). Completely washed, fixed cells were incubated with an anti-cleaved caspase-8 antibody (Cell Signaling) for 2 h, washed with PBS buffer, and incubated with goat anti-rabbit immunoglobulin G antibody conjugated with an FITC fluorescent dye (SantaCruz) for 1 h. Cells were then washed with PBS buffer and stained with DAPI (Sigma). Tunel staining was performed using the fluorometric TUNEL staining kit (Promega Corporation). In brief, cells were fixed and permeabilized with BD Cytofix/Cytoperm™ solution (BD biosciences). After washing, cells were treated with 50 μl of recombinant fluorescein-12-dUTP cocktail for 1 h at 37°C in a humidified chamber. Cells were then placed onto a slide glass with mounting medium with DAPI and the slides were sealed. Cells were examined in an LSM510 Meta confocal fluorescence microscope (Zeiss).

Mouse monoclonal anti-labyrinthin antibody MCA 44-3A6 was provided by the current lab [9 ] as grown using standard hybridoma technology and purified by 50% saturated ammonium sulphate precipitation and ion exchange chromatography or by a commercial kit (Pierce™ Antibody Clean-up Kit). Cytofix/Cytoperm solution, Perm/Wash buffer, biotinylated goat anti-mouse antibody, Fluorescein-5-isothiocyanate (FITC)-conjugated goat anti-mouse antibody, FITC-conjugated streptavidin, and propidium iodide were obtained from Becton Dickinson. BSA, trypan blue, and sodium azide were obtained from Sigma, and PBS (phosphate buffer saline) was purchased from Media Tech. Cell lines were obtained and grown as recommended by the American Type Culture Collection (Manassas, VA). Labyrinthin-negative WI-38 normal human lung fibroblast cells (negative control) and A549 human lung adenocarcinoma (positive control) cells were used in this experiment. Because adenocarcinomas are neoplasia of epithelial tissue that have a glandular origin, normal primary cultures of human astrocytes, renal proximal tubule epithelial cells, and small airway epithelial cells were selected as counter models in this study.

HL-1 cells were fixed and permeated with Cytofix/Cytoperm™ solution (Becton Dickinson, Franklin Lakes, NJ, USA), followed by washes with Perm/Wash™ buffer (Becton Dickinson, Franklin Lakes, NJ, USA). Non-specific antibody binding was blocked by incubation with 5% bovine serum albumin (BSA). Titin PEVK domain was stained with primary antibody 9D10 (Developmental Studies Hybridoma Bank at the University of Iowa, Iowa, USA) 1:100 diluted in 5% BSA, followed by secondary antibody Cy™5 (Invitrogen, Life Technologies, Grand Island, NY, USA) 1:100 diluted in 5% BSA. Nuclei were counterstained with DAPI. The patterns of titin staining were analyzed by Zeiss LSM 510 Confocal Microscope (Zeiss, Göttingen, Germany).
For determination of titin pattern in mouse heart tissue, 7 μm-thick cryosection slides of heart tissue were fixed and permeated with 0.2% PBST, and then blocked with 10% normal rabbit serum (NRS) in 0.2% PBST. Titin was stained with antibody 9D10 in 5% NRS and subsequently with the secondary antibody same as aforementioned. Confocal images were analyzed by software ImageJ (NIH, Bethesda, Maryland, USA).