Thermo shandon cryotome

Male and female PF23 H. contortus were extracted and fixed overnight in PBS 1X supplemented with 4% PFA at 4°C. The worms were washed 3 times in PBS 1X and incubated overnight at 4°C in PBS 1X with 30% sucrose under gentle rocking. The nematodes were rapidly frozen in dry ice after embedding in an optimal cutting temperature compound (OCT) (Thermo Fisher Scientific, USA). Cryosections were performed transversally using a Thermo Shandon cryotome (Thermo Fisher Scientific, USA). 10 μm slices were placed on microscope slides and blocked overnight in AbD. Worms sections were then independently incubated overnight at 4°C with a 1/100 dilution of each specific anti-Hco-UNC-29 antibody. After washing a minimum of five times with AbD at 4°C, the sections were incubated overnight with the corresponding 1:500 secondary antibodies conjugated to Alexa Fluor 488 (Invitrogen, USA). Final washes were performed two times in AbD and 2 times in 1X PBS/0.1% (v/v). Slides were then mounted using a Mounting medium (Sigma) and examined with the same confocal microscope.
The omission of the primary antibodies as well as the use of peptide adsorbed primary antibodies were used as controls. In the latter case, primary antibody were pre-adsorbed with 0.25 mg/ml of the corresponding peptide antigen.

Five μm sections of aortae were prepared using a Thermo Shandon Cryotome (Thermo Fisher Scientific, Waltham, MA, USA). The slides were kept frozen at −20 °C until stained using an Elastin Stain Kit (Sigma-Aldrich; St. Louis, MO, USA). The slides were fixed in 1% paraformaldehyde for 10 min, washed in 1× phosphate buffered saline (137 mM NaCl, 3 mM KCl, 10 mM Na₂HPO₄ and 1.5 mM KH₂PO₄) for 10 min, hydrated in double-distilled water for 15 min, and then stained according to the manufacturer’s protocol. Differentiation in the working ferric chloride solution was done for 3 min and staining in the Van Gieson solution was for 90 seconds. Slides were dehydrated in xylene, air-dried for 1 hour, and then mounted using VectaMount Permanent Mounting Medium. Slides were imaged with an EVOS^® microscope (Advanced Microscopy Group Solutions) under 4× and 20× objectives. ImageJ software was used to determine vessel morphology (4 × objective), and elastin and collagen content (stained black and red, respectively; 20 × objective) [16 (link)].

For assessing the size of DPC spheroids, DPCs (2x10⁵cells/well) were seeded in 24well Hydrocell plates (Nunc, Roskilde, Denmark), which have chemical coated surface for preventing cell attachment, consequently promoting spheroid formation. For immunocytochemical analysis, DPCs (1.5x10⁴cells/well) were seeded in 96well u-bottom hydrocell plate (Nunc, Roskilde, Denmark). Cells were incubated for 72 h to generate spheroids with / without PM extract. Then the size of spheroids was measured by microscopic photography (Leica, Wetzlar, Germany) and sorted by diameter.
For immunocytochemistry, 3D DP spheroids were embedded in OCT compound for cryo section. Then OCT compound embedded 3D DP spheroids were frozen at − 80 °C for more than 1 h. The frozen 3D DP spheroids were sliced in 15 μm thick using pre-chilled Thermo Shandon Cryotome (Thermofisher scientific, MA, USA).