Hm 325

Hepatic tissue samples were fixed directly in 10% neutral buffered formalin solution and embedded in paraffin wax. Paraffin blocks were cut to sections of 5 μm using a microtome (HM325, Thermo Fisher Scientific, Waltham, MA, USA). Hematoxylin-eosin (H&E) staining was performed according to the manufacturer’s guidelines (Merck Millipore, Darmstadt, Germany). Digital images of tissue slices were captured using a Zeiss microscope Mod. Axioplan 2 (Zeiss, Jena, Germany).
Oil Red O staining was performed on frozen liver sections (5 µm) previously fixed in 10% formalin for 5 min as previously described [60 (link)]. Briefly, slides were rinsed three times with absolute propylene glycol and then placed in 0.5% Oil Red O stain solution in propylene glycol for 30 min before being rinsed with 85% propylene glycol for 1 min and counterstained with hematoxylin. Thereafter, the slides were washed with distilled water and mounted with aqueous mounting medium (Sigma, St. Louis, MO, USA). Sections were observed with a Zeiss microscope Mod. Axioplan 2 (Zeiss, Jena, Germany).

Nodule fixed in 10% formalin following nodule extraction were processed manually, following the histopathology protocol from the Michigan State University, Department of Pathology/Histology; Issued by A. Porter, HT (ASCP) C and approved by William S. Spielman. Briefly, nodules were dehydrated by incubated in ascending graded strength of alcohol (60%, 75%, 80%, 96%) followed by clearing in 100% xylene then paraffin infiltration. The paraffin blocks containing nodules, were then cut in to 5 μm sections, using a hand held semi-automated micron rotary HM 325 (Thermo Fisher Scientific, Shangai, China). The tissues sections were then stained with hematoxylin and eosin (H&E) and the slides were mounted and observed under a light microscope (Human scope) using ×10 and ×40 objectives, for confirmation of specific morphological features of the worms, to appreciate live, dead and degenerated worms using a descriptive referenced by Büttner et al. (1988) .

H&E stain was performed as described earlier (70 (link)). Briefly, lungs were fixed in 10% buffered formalin and embedded in paraffin. Tissues were cut into 5 μm sections using a microtome (HM 325, Thermo Fisher Scientific). Sections were deparaffinized in xylene and then rehydrated. At least 3 independent tissue sections from each group were stained with H&E and examined for histological alterations using a Keyance microscope.
The immunohistochemistry was performed as described (64 (link)). PAD4 antibody (GeneTex, catalog GTX113945) at dilution 1:250 and anti–citrullinated histones (MilliporeSigma, catalog 07-596) at dilution 1:200 were used for this application.

The dorsal skin was resected, fixed in 10% formalin solution, and embedded in paraffin. The embedded specimens were then serially sectioned (5 μm) with a microtome (HM 325; Thermo Fisher Scientific) and stained with hematoxylin–eosin to observe the histopathological features or with Masson’s trichrome stain to examine the variable deposition of collagen fibers (blue) and skin fibrosis in the lesioned skin. Mast cells and eosinophils were stained with toluidine blue and Congo red, respectively. Additional information is provided in the Supplementary Materials and Methods.

Samples were prepared from maxillae for histological analyses. After fixing the samples with 4% paraformaldehyde for 24 hours, decalcification solution (Yobibio, YB2003) was used for demineralization at 4°C, and the solution was changed every two days. Paraffin embedding was carried out one month later. Paraffin-embedded blocks were sectioned at 5μm intervals using a microtome (HM325, Thermo Fisher Scientific, USA). Ly6G⁺ cells in the mouse maxillae were examined by immunohistochemistry (IHC) with a the specific primary antibody (1:500; Abcam, ab238132) and a diaminobezidin (DAB) system (Absin, abs957) according to the manufacture’s instruction. Five high-power fields (hpf) (×400) per each sample at ROI were randomly selected and the positively-stained cells were enumerated by Image Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD).

Following transcardial perfusion in 1× PBS, left brain hemispheres were fixed in 4% paraformaldehyde overnight (4°C) and processed using an Excelsior tissue processor (Thermo Fisher Scientific). Samples were then embedded in paraffin and sectioned at a thickness of 3 μm (Thermo Fisher Scientific, HM325). Immunostaining was performed as previously described (10 (link)). Briefly, following antigen retrieval, histological sections were blocked and permeabilized (5% donkey serum + 0.1% Triton X-100). Primary antibodies, anti-GFP (Abcam, ab290) and anti-HA (Cell Signaling Technology, clone C29F4: 3724S), to visualize AAV-transgene expression, were incubated on slides overnight (4°C). Alexa fluorophore-coupled secondary antibodies 488 and 568 (Molecular Probes) diluted in blocking buffer (5% donkey serum) were incubated on slides at room temperature for 1 hour before 4′,6-diamidino-2-phenylindole staining for 10 min. Slides were mounted for confocal imaging using an AxioScan microscope slide scanner (Zeiss).