Bsa pbs

The samples were electrophoresed in 4–12% Bis-Tris gels at 100 V using MES running buffer. The proteins were electrophoretically transferred to a nitrocellulose membrane at 100 V for 1.5 h. The membrane was blocked with 3% milk in PBS 1X at 37°C for 2 h. The blots were incubated with primary antibodies: rabbit anti human LASP-1 polyclonal antibody, 1:500 in 0.3% BSA-PBS (Millipore); rabbit anti-green fluorescent protein polyclonal antibody, 1:100 in 0.3% BSA-PBS (Santa Cruz Biotechnology); mouse anti-vimentin monoclonal antibody, 1:1000 in 0.3% BSA-PBS (Santa Cruz Biotechnology) at room temperature overnight, washed three times with PBS, then incubated with an alkaline-phosphatase-conjugated secondary antibody (1:7500 in 0.3% BSA-PBS) for 4 h at 37°C. The results of the immunoreaction were detected with Nitroblue tetrazolium and bromochloroindolyl phosphate (Promega).

Immunofluorescence staining was used to determine the effect of vancomycin on the differentiation of the isolated muscle cells. Therefore cells were cultured in collagen (Corning^®; Discovery Labware; Amsterdam; Netherlands) coated 24 well plates. After reaching 80% confluency the medium was replaced by differentiation medium including vancomycin in different concentrations. Five days later, cells were washed twice with PBS, followed by fixation with ice cold methanol (Carl-Roth^® GmbH, Karlsruhe, Germany) for 20 min. After a second washing step with PBS and 20 minutes incubation with the primary antibody skeletal muscle myosin (F59) anti mouse (1:200 in 0,5 % BSA/PBS; Santa Cruz Biotechnology, Texas, TX., USA) was added and incubated at 4 °C overnight. As secondary antibody Alexa 488 goat anti mouse IgG (1:200; Invitrogen™Life Technologies, Carlsbad, USA) was applied for 1 h in the dark followed by washing with PBS. Finally nuclei were stained with Hoechst (4 µg/mL; Sigma Aldrich, St. Louis, MO, USA) with an incubation time of 15 minutes at room temperature in the dark. Detection was performed with the EVOS^® digital Inverted Microscope (Life Technologies, Carlsbad, CA, USA).

A549 cells were grown and treated on glass slides then fixed and permeabilized with 70% acetone/30% methanol solution at −20°C for 10 minutes. The slides were air-dried, blocked with 10% donkey serum in PBS (60 min) (Jackson Immuno Research Laboratories, West Grove, PA, USA), and incubated overnight incubation at 4°C with (1∶50) rabbit polyclonal anti-p65 (NF-κB) antibody in 1% BSA/PBS (Santa-Cruz Biotechnology Inc, Santa Cruz, CA, USA). After washing, the slides were incubated for 1 hour at room temperature with (1∶100) Alexa fluor 488 conjugated donkey anti-rabbit antibody, and Alexa fluor 647 conjugated WGA to stain the membranes ((Molecular Probes Invitrogen Detection Technologies, Eugene, OR, USA). The chromatin stain DAPI was employed to demarcate the nuclei (Sigma-Aldrich, St. Louis, MO, USA). Images were acquired with a Leica DMRXA fitted with a Cooke CCD SensiCam using Chroma Sedat filters with single excitation and emission filter cubes. The three channel images were then digitally processed, for calculations of fluorescent mean intensities and for mean cell size (in voxels) measurements, using Intelligent Imaging Innovations Slidebook 4.1 software (Intelligent Imaging Innovations Inc, Denver, CO, USA).