Anti vimentin

For immunolabeling, primary fibroblasts were seeded and grown on chamber slides (ThermoFisher Scientific) for 24 h, fixed in 4% paraformaldehyde (Sigma-Aldrich), blocked in 10% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, USA), then incubated overnight (at 4 °C) with anti-α-SMA, anti-vimentin (Novus Biologicals, Abingdon, UK), anti-TGF-β, and collagen 1 (Abcam, Hong Kong, China). Heart sections were incubated at 4 °C overnight with anti-α-SMA, anti-vimentin (Novus Biologicals), and anti-α-sarcomeric actin (Sigma-Aldrich). The primary antibody was revealed using respective anti-mouse IgG/IgM, anti-rabbit IgG, or anti-chicken IgG secondary antibody (Jackson ImmunoResearch). The nuclei were visualized via DAPI staining [12 (link)]. Samples were analyzed using a confocal microscope (LSM700, Zeiss, Jena, Germany).

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.

Primary trophoblast cells were isolated from healthy term placental villous tissues. Placental tissue was carefully minced, and approximately 30 g was digested three times for 30 min at 37°C in saline Hanks/HEPES solution including DNase I (Sigma‐Aldrich, USA) and trypsin (Thermo Fisher Scientific, USA) as previously described.
⁴¹ (link) After digestion and centrifugation at 1000 RCF, the cellular pellets were resuspended in DMEM and separated by centrifugation (1500 RCF, 20°C, 20 min) using a 10%–70% Percoll gradient (Sigma‐Aldrich, USA). Trophoblast cells were obtained from Percoll gradient fractions between 35% and 55%. Cells were plated at a density of 200 000 cells/cm² in DMEM high‐glucose (4.5 g/L glucose) medium supplemented with 10% fetal bovine serum and 1x antibiotic–antimycotic (Gibco, USA). After isolation, the cells were cultured for 12 h to allow them to attach before being exposed to different oxygen conditions.
The purity of the isolated trophoblast was evaluated by staining with the specific cell markers anti‐cytokeratin 7 (CK7), anti‐vimentin, or anti‐von Willebrand factor (vWF) (Novus Biologicals). Cells were acquired by flow cytometry (BD FACS LSRII; BD Biosciences, USA) as described.
³⁸ (link) The purity of the isolated trophoblasts was 91%–96%.