Mouse embryonic fibroblasts were derived from wild-type and vimentin null mice and immortalized by stable expression of SV40 large T-antigen (kindly provided by J. Ericsson, Abo Akademi University, Turku, Finland)25 (link). Cells were grown in 1X DMEM (Life Technologies; Catalog no: MT10013CV) supplemented with 10% fetal bovine serum (GE Healthcare Life Sciences, Catalog no: SH3008803), 1% penicillin-streptomycin (Gibco) and nonessential amino acid (Life Technologies), and 10 mM HEPES and sodium pyruvate (Life Technologies) at 37 °C with 5% CO2. Cells were plated at a density of 10,000 cells/gel or less.
For immunofluorescence experiments, cells were fixed with 4% paraformaldehyde (Affymetrix) followed by 5% BSA and 1% Saponin (Sigma) for blocking and permeabilization. Primary antibodies were Alexa- Fluor 647 phalloidin (Invitrogen Catalog no: A22287), anti-vimentin (Novus Biologicals Catalog no: NB300-223), and dapi (Sigma Catalog no: D9542). Alpha-tubulin rat antibody (Bio-Rad- Catalog no: MCA77G) at a concentration of 1:200 was used for detecting microtubules.
For immunofluorescence experiments, cells were fixed with 4% paraformaldehyde (Affymetrix) followed by 5% BSA and 1% Saponin (Sigma) for blocking and permeabilization. Primary antibodies were Alexa- Fluor 647 phalloidin (Invitrogen Catalog no: A22287), anti-vimentin (Novus Biologicals Catalog no: NB300-223), and dapi (Sigma Catalog no: D9542). Alpha-tubulin rat antibody (Bio-Rad- Catalog no: MCA77G) at a concentration of 1:200 was used for detecting microtubules.
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