Hematoxylin

Fixed samples were decalcified in 20% EDTA (pH 7.5), embedded in paraffin and cut into 5 μm sections using an HM360 microtome (Microm). Hematoxylin (VWR) and eosin (Sigma–Aldrich) staining was performed to assess histomorphology. Tartrate-resistant acid phosphatase (TRAP) (Sigma) was used to detect osteoclasts according to the manufacturers’ protocols. For immunofluorescence staining, slides were subjected to sodium citrate buffer at 95 °C for 20 min for antigen retrieval, permeabilized with 0.5% Triton X-100 (Beyotime) for 10 min, and blocked with 5% BSA for 1 h. Slides were incubated with Anti-Runx2 (1:200, Abcam, ab23981), Anti-GFP (1:50, Santa Cruz, sc-9996), Anti-RFP (1:50, Santa Cruz, sc-390909), Anti-Klotho (1:100, R&D, AF1819), Anti-Klotho (1:100, Santa Cruz, sc-515939), or Anti-Rankl (1:100, R&D, AF462) antibody overnight at 4 °C, and a fluorescence-conjugated secondary antibody, Alexa Fluor 488 or 568 (Invitrogen, 1:1000) for 1 h at room temperature. Nuclei were counterstained with DAPI (Vector). Images were captured on an Olympus confocal microscope FV3000 (Olympus).

Treatment- and test-naive C57BL/6J males and females were used. Mice received a single dose of Raphin1 or cycloheximide (CHX, Sigma-Aldrich) at 40 mg/kg at 3 months of age or were treated chronically with Raphin1 at 2 mg/kg from 4 weeks of age for 10 weeks before the analysis. Mice were culled by cervical dislocation, liver was extracted, fresh-frozen in the O.C.T. compound (VWR International) and kept at −80°C until use. Oil Red O staining was performed on 10 μm thick frozen cryosections (Leica CM1850). Sections were washed in 60% propanol and stained for 20 min at room temperature with filtered 0.33% Oil Red O solution (VWR International). After rinsing with distilled water sections were counterstained with hematoxylin (VWR, diluted 1:15) for 1 min. Images were acquired with a 40X objective using SCN400F scanner (Leica).

Sections were fixed in Bouin’s fluid, dehydrated in graded dilutions of ethanols (70–100%) and embedded in paraffin. For staining, 3 μm sections were cut and mounted on Superfrost Plus slides (Thermo Scientific, Dublin, Ireland). Cryosections were prepared as follows: testicular tissue samples (1 cm) were snap-frozen in liquid nitrogen immediately after removal. After cryotomy, sections (8 μm) were put on coated slides (Superfrost Plus, Menzel, Braunschweig, Germany) and dried at room temperature. The slides were frozen at −20 °C until use.
hematoxylin and Eosin (H&E) staining was carried out to investigate testicular morphology. Following dewaxing in xylene (Sigma-Aldrich, Wicklow, Ireland; room temperature [RT], 3 × 10 minutes [min]), rehydration in descending alcohols (RT, 100% 5 min, 100% 5 min, 80% 5 min, 70% 5 min) and distilled water (RT, 5 min), sections were put in hematoxylin (VWR, Dublin, Ireland; RT, 30 seconds [s]), running water (15 min), eosin (Sigma-Aldrich, Wicklow, Ireland; RT, 45 s) and then running water (5 min). Sections were then dehydrated in ascending alcohol solutions (RT, 70% 5 min, 80% 5 min, 100% 5 min, 100% 5 min), cleared in xylene (Sigma-Aldrich, Wicklow, Ireland; RT, 3 × 10 min), and mounted in DPX (Sigma-Aldrich, Wicklow, Ireland).

Haemonchus contortus adult worms were collected from the abomasa of infected animals, fixed in 10% neutral formalin, embedded in paraffin and sectioned at 5 µm thickness (HistoServe Inc., Germantown, MD, USA). The slides were deparaffinized and rehydrated through xylene/ethanol washes, then quenched with 3% H₂O₂ and rehydrated prior to antigen recovery in 0.4% pepsin 1% hydrochloric acid at 37 °C for 15 min. The sections were washed twice with 0.75% of 30% BRIJ-35 (Millipore Sigma, Burlington, MA) in 1× PBS (BRIJ–PBS), blocked with 0.5% sodium caseinate in BRIJ–PBS for 10 min and then incubated with rabbit antisera to rHc-CBP-1 or rHc-CBP-2 (1:800 dilution) and pre-immune sera (1:800 dilution) for 30 min at room temperature. After washing, the slides were incubated with goat anti-rabbit HRP conjugated secondary antibody (GARP) for 20 min and treated with 3,3' diaminobenzidine (DAB) substrate (Abcam, Cambridge, MA). Hematoxylin and HistoMark TrueBlue (VWR, Radnor, PA) were used as counterstains. Micrographs were generated using the Zeiss Axioskiope 2 Plus microscope (Zeiss, Thornwood, NY, USA).

The following reagents were used during the course of the experiments and were purchased as following: red blood cell (RBC) lysis buffer was purchased from Millipore Sigma (Burlington, MA); Percoll from GE Healthcare Life Sciences (Pittsburgh, PA); Myelin oligodendrocyte glycoprotein peptide subunit 35–55 (MOG35-55) from PolyPeptide Laboratories (San Diego, CA); Pertussis Toxin (PTX) from List Biological Laboratories (Campbell, CA); Heat killed Mycobacterium tuberculosis (H37Ra) from Difco (Detroit, MI); β-mercaptoethanol, Freund’s Advjuvant, propionic acid, n-butyric acid, isovaleric acid, 2-ethylbutyric acid, sulfuric acid and Tween-80 from Sigma-Aldrich (St. Louis, MO); absolute ethanol, acetic acid, lithium carbonate and tryptamine were purchased from Fisher Scientific (Hamptom, NH); RPMI 1640, fetal bovine serum (FBS), L- glutamine, phosphate buffered saline (PBS), luxol fast blue (LFB), cresyl violet, eosin, hematoxylin, and HEPES were purchased from VWR (West Chester, PA; flourophore conjugated antibodies and ELISA kits from Biolegend (San Diego, CA); Illumina MiSeq reagents from Illumina Inc. (San Diego, CA); QIAamp Stool Mini Kits from Qiagen (Germantown, MD); 10% formalin from Azer Scientific (Morgantown, PA).