Axioscop 2

Control and CIH guinea pig CB were perfused and fixed as described in Gonzalez-Obeso et al. (2017) (link). CB were cryoprotected by immersion in 30% (w/v) sucrose in phosphate buffer, embedded individually in Tissue-Tek^® (Sakura Finetek, Zoeterwoude, Netherlands) and frozen at -20°C. Tissue sections of 10 μm (Shandon Cryotome, Thermo, Electron Corporation) were collected in glass slices coated with poly L-lysine. CB sections were washed with PBS at room temperature, hematoxylin and eosin stained (H&E, Sigma-Aldrich, MO, United States), dehydrated and mounted with Eukitt Mounting Medium (Merck). Tyrosine hydroxylase (TH) immunofluorescence staining was performed in slices from control and CIH CB, identifying cell nuclei with DAPI. Sections were examined with microscope (Axioscop 2, Zeiss). Images were captured using a digital camera (CoolSNAP, Photometric, Roper Scientific) coupled to the microscope and analyzed using Metamorph 6.3 software.
Dissociated CB cells (Gomez-Niño et al., 2009 (link)) from four different control and four different CIH animals plated on several poly L-lysine-coated coverslips were immunostained for TH and nuclei with DAPI as previously described (Caceres et al., 2007 (link); Gonzalez-Obeso et al., 2017 (link)). Cells were imaged using a laser confocal microscope (LEICA TCS, SP5) and confocal micrographs were processed using LAS software.

Mammary tissue was formalin fixed (4% v/v), paraffin embedded before sectioning (5 μm), and subjected to standard H&E staining. Histology was imaged with a Zeiss Axioscop2 microscope using PlanNeoFluar 40× lens (numerical aperture 0.75) fitted with a Zeiss AxioCam color camera, and analyzed with Openlab (3.1.7) software (Improvision).
Whole-mount analysis was performed by spreading inguinal mammary glands on polysine slides and stained with carmine alum as described previously (Akhtar et al., 2009 (link)).
ORO staining was performed by staining 10 μm mammary cryosections in freshly diluted ORO solution (6 parts 0.5% ORO stock solution [Sigma] and 4 parts water) for 15 min. Sections were rinsed twice with 60% isopropanol, once with water, and then counterstained with Mayer's hematoxylin for 1 min before photography as above.

All nematodes were either viewed on plates using a Zeiss bench-top microscope fitted with a Canon Sureshot camera or were mounted on 2% agar pads on slides and viewed under Differential Interference Contrast (DIC) or fluorescence optics on a Zeiss Axioscop2 and imaged with a Zeiss AxioCam camera and Axiovision software.

Dog serum samples were tested with a commercial indirect fluorescent antibody test (IFAT) (MegaScreen®FLUO Babesia canis, MegaCor Diagnostic, Horbranz, Austria) to detect the presence of IgG antibodies against B. canis, using canine erythrocytes infected with B. canis as antigens. Positive and negative controls were always included and were provided by the company. Starting, sera were initially diluted 1:64 in phosphate-buffered saline solution (PBS) (pH = 7.2), applied to slide wells and incubated in a moist chamber for 30 min a 37 °C. The slides were washed and reacted with fluorescein isothiocyanate-conjugated rabbit anti-Dog IgG (Sigma-Aldrich, Milan, Italy), incubated at 37 °C in a humid chamber for 30 min. The slides were mounted with buffered glycerin and covered with a coverslip. Finally, the slides were examined with a microscope (Axioscop 2, Zeiss) under a fluorescent light (HBO50). Those samples that had fluorescence at a diluition ≥ 1:64 were considered as seropositive. Samples resulting seropositive were then diluted to determine the end-point titre (i.e. the last dilution in which is observed positive reaction).

Primary human corneal stromal cells were established from a donor cornea that was not suitable for transplantation. The primary cells were maintained in SF-1 hMSC medium (United Healthcare Inc., Taiwan). Full-length mouse Wnt5a cDNA (pLNC Wnt-5aHA) was a gift from Jan Kitajewski (Addgene plasmid #18032) [17 (link)], and mouse sFRP1 cDNA XE141 sFRP-1-CS2+ was a gift from Randall Moon (Addgene plasmid #16693; http://n2t.net/addgene:16693; RRID : Addgene_16693). These cDNA constructs were used to generate the recombinant adenovirus vector according to a published protocol [18 (link)]. Another recombinant adenovirus coding GFP alone (AdGFP) was used as a control. AdGFP, AdsFRP1, and AdWnt5a viruses were amplified and purified according to the protocol.
Cells were seeded into Corning™ 96-Well Half Area High Content Imaging Film Bottom Microplate and were infected with AdGFP or AdsFRP1 after 24 hours. 72 hours after infection, the cells were fixed for 20 minutes in 1% glutaraldehyde. Cells were permeabilized with 0.2% Triton X-100 in PBS and were stained with an antibody against sFRP1 (Novus Biologicals, NBP1-02432), CHOP (Cell Signaling, 5554S), and ROR2 (Cell Signaling, 88639S). The images were taken with an epifluorescence microscope (Axioscop2, Carl Zeiss, München-Hallbergmoos, Germany) and were photographed with a digital camera system (Axiocam, Carl Zeiss).