Entellan

c-FOS was detected using a rabbit polyclonal antibody against c-FOS (sc-253 Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; 1:8000; 48 h; 4°C) according to the same protocol as above. The free-floating sections were then incubated with either a mouse polyclonal antibody against TH (MAB318, Millipore, 1:4000) or a rabbit polyclonal antibody against 5-HT (S5545, Sigma–Aldrich, Saint-Quentin Fallavier, France; 1:500; 48 h; 4°C). Sections were subsequently incubated for 2 h with biotinylated horse anti-mouse (Vector Laboratories, Burlington, Canada; 1:500) or goat anti-rabbit (Vector Laboratories, Burlington, Canada; 1:500) antibodies, and then with an avidin-biotin-peroxidase complex (1:250). The TH and 5-HT immunoreactivities were detected by incubation for 3–5 min in NovaRED (Vector Laboratories, Burlington, Canada).
Sections were mounted in sequential caudo-rostral order on silane-treated slides as for the single immunohistochemical detection of c-FOS. They were air-dried, dehydrated with absolute alcohol, cleared with xylene, and coverslipped using Entellan® (VWR International S.A.S).

Native mite sections were stained with the modified hematoxylin-eosin (H&E) fast staining kit (Carl Roth, Karlsruhe, Germany) according to the manufacturer’s protocol. Briefly, sections were stained with H&E solution 1 for 6 min and rinsed with tap water for 10 s. Subsequently, the tissue was covered with 0.1% hydrochloric acid, and after incubation for 10 s, the slide was rinsed with tap water for 6 min. H&E solution 2 was added to the slides and was removed after 30 s by rinsing with tap water for another 30 s. Stained mite sections were air dried and covered with a cover slip by using Entellan (VWR, Darmstadt, Germany).

Liver slides (paraffin sections 2µm) were deparaffinized using Roti^®-Histol (Carl Roth, 6640) and rehydrated by ethanol gradient (100–40%). H&E staining was performed by firstly adding hematoxylin solution (Sigma-Aldrich, GHS316, MerckKGaA, Darmstadt, Deutschland) for 45 s followed by 10 s tap water and incubation of eosin (Sigma-Aldrich, HT110232, MerckKGaA, Darmstadt, Deutschland) for 1 min. After staining, samples were mounted with Entellan^® (VWR, 1079610500, MerckKGaA, Darmstadt, Deutschland). Liver sections were scanned using a MIRAX Scanner from Zeiss and software MIRAX Viewer.

For staining of human plaques, IHC reagents were from Biocare Medical, Pacheco, CA, USA. Isotype rabbit and mouse IgG were used as negative controls. In brief, 5 μm sections were deparaffinized in Tissue Clear and rehydrated in ethanol. For antigen retrieval, slides were subjected to high-pressure boiling in DIVA buffer (pH 6.0) or TE buffer (pH 9.0). After blocking with Background Sniper, primary antibodies diluted in a Da Vinci Green solution were applied and incubated at room temperature for 1 h. A double-stain probe-polymer detection kit (Mach 2) containing both alkaline phosphatase and horseradish peroxidase was applied, with subsequent detection using Warp Red and Vina Green. All slides were counterstained with hematoxylin (HTX QS, H-3404, Vector Laboratories Inc., Burlingame, CA, USA), dehydrated and mounted in Pertex (Histolab, Gothenburg, Sweden). Images were taken using a Nikon Eclipse E800 microscope.
For staining of mouse sections, sequential 5 μm slides were rehydrated, antigens were retrieved by boiling in a TriSodiumCitrate buffer (pH 6.0). Primary antibodies were visualized with a Nova-RED substrate (Vector #SK-4800, Vector Laboratories Inc., Burlingame, CA, USA). Sections were counterstained with hematoxylin (#4085-9002, Klinipath, VWR, Radnor, PA, USA) and mounted with entellan (#7961, Burlington, MA, USA).

CO₂/H⁺-activated cells of the RTN/pFRG are described as PHOX2B-positive (Mulkey et al., 2004 (link); Guyenet et al., 2013 (link); Kumar et al., 2015 (link); Ruffault et al., 2015 (link)). To compare distribution of PHOX2B-positive neurons in RTN/pFRG between WT and Epo-TAg^h mice, sections were incubated with a polyclonal goat anti-PHOX2B antibody (sc-13226; Santa Cruz Biotechnology Inc., United States; 1:750) in 1% bovine serum albumin for 48 h at 4°C. Afterward, sections were firstly incubated with a biotinylated horse anti-goat immunoglobulin (BA-9500; Vector Laboratories, Canada; 1:500) in 1% bovine serum albumin for 2 h, and then with ABC in 1% bovine serum albumin for 1 h, respectively. Peroxidase activity was detected with 0.02% 3,3′-diaminobenzidine tetrahydrochloride and 0.01% H₂O₂ in 0.005 M Tris-HCL buffer. Sections were mounted in sequential caudo-rostral order on slides, air-dried, dehydrated with absolute alcohol, cleared with xylene and coverslipped with mounting medium (Entellan^®, VWR International S.A.S).