Molecular biology studies

All experimental procedures concerning this chapter were already described in full detail (Milcheva et al., 2023). In brief, the expression of mRNA of mouse Nitric oxide synthase 1 (Nos1) was evaluated by real-time RT-PCR in tissue samples from mouse skeletal muscle collected at 0, 14, 24, and 35 d.p.i. The levels of expressions were estimated via normalization versus the expression of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) as a reference gene. The infection of the samples was confirmed by end-point PCR of the Expansion Segment V (ESV) of T. spiralis (Zarlenga et al., 2001), as already described in detail (Milcheva et al., 2023). Briefly, genomic DNA from six paraffin sections from all samples was isolated using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden Germany). Genomic DNA from T. spiralis infectious larvae was isolated as a positive control, using NZY Tissue gDNA Isolation Kit (NZYTech, Lisboa, Portugal). All isolations were performed according to the provided protocols of the producers. The yield and purity of the collected gDNA were measured using S-300 Spectrophotometer (Boeco, Hamburg, Germany). Hot start PCR was designed on approximately 100 ng gDNA as a template by using Veriti thermoblock (Applied Biosystems of Thermo Fisher Scientific) under standard PCR conditions (Milcheva et al., 2019). The products of amplification were visualized on 2.5 % agarose gel supplemented with Simply Safe nucleic acid stain (EurX®, Gdansk, Poland) versus 100-1000 bp DNA Ladder (EurX), and the gels were photographed with a gel documentation system Vision (Scie-Plas Ltd, Cambridge, UK). The primers used for gene expression analyzes were designed using the NCBI Blast Tool (Ye et al., 2012) in a way to span at least one intron sequence. The full names of investigated genes, the accession numbers of their reference sequences, the primer sequences, and the size of the amplified products are shown in Table 1. The oligonucleotides were purchased from HVD BiotechVertriebs (Vienna, Austria).

The full names of the investigated genes and their primers sequences used in this study.