Las version 3

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).

Germline proliferation-deficient worms (Glp) were produced as previously described17 (link) to avoid difficulty in GFP observation within transgenic worms (ugt–29::GFP, hsp–16.2::GFP and pgp–5::GFP) due to the spatial interference by nematode eggs. For every indicated experimental condition, ~ 100 Glp transgenic worms were transferred to the assay plates (pathogen-seeded, RNAi or chemical–supplemented plates) and kept at 25 °C until the completion of the assay. At every indicated time point, 10 to 15 transgenic worms were mounted on a 2% agarose pad prepared on a glass slide for GFP examination. Five microliter of 5 mM levamisole was used as the paralyzing agent. The worms were observed under 100× and 400× magnification using a Leica upright fluorescence microscope equipped with a Leica I3 long pass GFP filter (Leica Microsystems) and all micrographs were captured using the Leica DCF 310 FX digital camera and LAS version 3.8 software (Leica Microsystems).