E0466

The first part of the IF protocol was carried out as described for IHC in Supplementary Table S4. After being processed as previously described [3 (link)], the slides were incubated for 30 minutes at room temperature (RT) with rabbit anti-goat biotinylated antibody (E0466 DAKO, Carpinteria, CA) (dilution 1:100), followed by Alexa Fluor 546 streptavidin for one hour at RT (dilution 1:1000) (Invitrogen, Carlsbad, CA). Thereafter, the slides already incubated with anti-gremlin or VEGFR2 antibody (Supplementary Table S4, part 2), were incubated with Alexa Fluor 488 anti-rabbit antibody (Invitrogen) (dilution 1:1000) for one hour at RT. The rest of the protocol is described in Supplementary Methods.

Samples were taken from the liver, left lung, and MLN on 14 and 31 dpi. Tissues were fixed in 4% formaldehyde in phosphate-buffered saline. After dehydration, samples were embedded in paraffin, sectioned at 2–4 µm and stained with haematoxylin and eosin (HE). Eosinophilia was categorised after the number of eosinophils: none-mild (< 50 per high-power field) or moderate-massive (≥ 50). To facilitate the evaluation of fibrosis, selected slides were stained for connective tissue by the Masson trichrome (MT) technique [38 ], and fibrosis was graded as nil-mild, moderate, or massive, as defined in Supplemental Fig. 1-2. Selected samples were immunohistochemically stained for ionised calcium-binding adaptor molecule 1 (IBA1) to identify macrophages and facilitate evaluation of the inflammatory response. An avidin/biotin complex (ABC) method was used, where non-specific binding cites were blocked with 4% normal rabbit serum (X0902; Dako, DK), the primary antibody was a polyclonal goat anti-IBA1 (ab5076; Abcam, UK), and the secondary antibody was a biotinylated rabbit anti-goat (E0466; Dako, DK) [39 ].
In Exp. 1, standard haematological analyses were performed using an ADVIA2120 haematology analyser (Siemens), including white blood cells (WBC) and eosinophils (EOS).

Brains or embryos were dissected in phosphate buffered saline (PBS), fixed overnight in 4% paraformaldehyde (PFA) at 4°C, dehydrated and embedded in paraffin wax. Serial, sagittal sections were cut at 10 μm and left to dry overnight at 42°C. Sections were stained with Cresyl Violet or processed for immunohistochemistry as described (Whittaker et al., 2017 (link)). The following primary antibodies were used: anti-tyrosine hydroxylase (Abcam, ab112; 1:200) and anti-Neurogranin (Millipore, AB5620; 1:500). Primary antibodies were detected using Alexa fluor-conjugated secondary antibodies (Invitrogen; 1:200) or biotinylated secondary antibodies (Dako, E0466; 1:200) with the Vectastain ABC Kit (Vector Laboratories) and visualized using 0.03% diaminobenzidine (DAB; Sigma).