Vimentin

Frozen tissue sections of 5 μm thickness were fixed with 4% formalin and subjected to histological and immunohistological staining. Hematoxylin and eosin (H&E) staining was assessed for orientation and tissue integrity and Movat pentachrome staining was used to assess distribution of collagen, glycosaminoglycans, and elastic fibers within the valvular tissue. Biomineralization was confirmed via Alizarin red and von Kossa staining. Staining procedures were performed as previously described [20 (link),21 (link)].
Immunohistological staining was performed as previously described [22 (link)] with antibodies targeting vimentin (Progen Biotechnik GmbH, Heidelberg, Germany) and von Willebrand factor (vWF; Agilent Technologies, Santa Clara, CA, USA). Subsequently, sections were stained with alexa488 and alexa594 conjugated secondary antibodies (Thermo Fisher Scientific). Sections were embedded with Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA). Images of all sections were taken using a Leica DM 2000 and LAS version 3.8 software (Leica Microsystems, Wetzlar, Germany).

Cells were fixed in 3.8% (w/v) formaldehyde for 10 min at room temperature, and blocked with 10% (v/v) FBS in PBS containing 0.1% (v/v) Triton X-100 for 1 h at room temperature. Cells were incubated with primary antibodies overnight at 4 °C. The primary antibodies used were: anti-neuron-specific class III beta-tubulin (TUBB3, TUJ1, 1:7500, Covance Inc. Princeton, NJ, USA), microtubule-associated protein 2 (MAP2; 1:500, Millipore, Bedford, MA, USA), SMI32 (1:1000, Covance Inc. Princeton, NJ, USA), ISL1/2 (39.4D5, 1:5, developed by T.M. Jessell and S. Brenner-Morton was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242), S100β (1:200) and vimentin (1:1000 Progen, Heidelberg, Germany). The following day, coverslips were washed and incubated with Alexa-Fluor conjugated secondary antibodies (1:1000) for 1 h at room temperature, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 0.3 µM). Cells were mounted in Aqua-Polymount (Polysciences, Inc., Warrington, PA, USA).