S 1000

Any lung sample categorised as ‘MSO’ on gross examination, ‘HSO’ on histological examination, and/or any that yielded a positive RT-PCR result (total: n = 46) were subjected to IHC using a primary antibody against the SU sub-unit of the envelope protein of JSRV, as described previously [29 (link)]. Tissue from one sheep that was MSO-positive (Sheep 46) was unavailable for IHC. Briefly, sections were dewaxed and rehydrated to water. Endogenous peroxidase activity was blocked by incubation in 3% H₂O₂ in methanol (v/v) for 20 min, then washed in running tap water for five minutes. Slides were then placed in citrate solution, pH 6.0, at 121 °C for 10 min for antigen retrieval, washed, and non-specific antibody binding was blocked by incubation with 25% normal goat serum (Vector S1000) diluted in phosphate buffered saline containing 0.05% Tween₂₀ (PBST) for 30 min prior to incubation overnight with the primary antibody (mouse anti-SU protein, diluted 1/250 in PBST). After washing, bound primary antibody was visualised with the DAKO EnVision™ + System (goat anti-mouse, DAKO) as per the manufacturers’ instructions using 3, 3′-diaminobenzidine as a substrate (Sigma-Aldrich). Sections were counterstained with Haematoxylin Z, blued in Scots tap-water substitute, dehydrated, cleared and mounted. Slides were washed in PBST between procedures unless otherwise stated.

For diffusion experiments (Fig. 1), immunostaining was done on free-floating brain sections using the ABC method. Free-floating sections were rinsed with TBS and heated in 10 mM citrate buffer, pH 6, with 0.05% Tween 20 at + 80 °C for 30 min, after they were blocked with 2% normal goat serum (S1000, Vector) in TBS-T (0.015% serum in TBS-T) for 20 min. Sections were then incubated in rabbit anti-human CDNF antibody (stock solution 0.4 mg/ml, Icosagen, 1:500) overnight at + 4 °C. Sections were rinsed in TBS-T (TBS, 0.1% Tween 20) and incubated in biotinylated goat anti-rabbit IgG (1:200, Vector Laboratories, Burlingame, CA) for 1 h in the room temperature, followed by incubation for 30 min with avidin–biotin-horseradish peroxidase complex (ABC kit; Vector) and lastly reacted with 40 s–1 min with 3,3′-diaminobenzidine tetrahydrochloride (DAB) (SK-4100, Vector). Sections were then mounted on slides with coverslips.

Paraffin-fixed sagittal brain sections were processed for Aβ immunohistochemistry. Sections were boiled in citrate buffer (pH 6.0, 10 mM trisodium citrate) for 5 min followed by cooling. After blocking with 5% goat serum (S-1000, Vector Laboratories Inc) for 30 min, brain sections were incubated with an anti-82E1 (1 : 100, 10323, IBL) antibody overnight at 4°C followed by incubation with goat anti-mouse Alexa Fluor 488 (A11029, Thermo Fisher Scientific) for 1 h at room temperature. Nuclei were stained with DAPI (P36931, Vector Laboratories Inc). Images were obtained using a microscope (Carl Zeiss). Aβ plaques were evaluated as the percentage of the immunostained area (positive pixels) divided by the total area examined (total pixels) using ImageJ software.

A dual-label confocal immunofluorescence examination was used to observe the potential crosstalk between substance P and ERβ in the spinal cord (Li et al., 2013 (link)). By boiling in 0.01 M sodium citrate buffer, pH 6.0, slides were blocked with normal 5% goat serum (Vector, S-1000). They were incubated overnight with a mouse anti-substance P antibody (Santa Cruz, sc-58591, 1:100 dilution). Then at room temperature, slides were incubated with Alexa Fluor^§ 594 Goat anti-rabbit (Invitrogen, A11037) for 30 min in the dark. Three times washing in phosphate-buffered saline (PBS) for 5 min each. Room temperature incubation with rabbit anti-ERβ antibody (Affinity, AF6469, 1:100 dilution) for 30 min in the dark, followed by 30 min of fluorescent conjugated secondary antibodies of Alexa Fluor^§ 488 Goat Anti-Rabbit IgG (Invitrogen, Cat:A11034), rinsed three times with PBS. The final step involves mounting slides in the fluorescent mounting medium containing 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen, P36935). All slides were viewed with a microscope (Nikon Eclipse 50i). Nucleus was visualized as blue by 330–380 nm filter, green mark with 465–495 nm filter, and red mark with 530–600 nm filter.

After plating of hepatocytes extracted from Bsg^+/+ or Bsg^–/– mice, preparation for immunocytochemistry was performed as described previously (45 (link)). In brief, samples were fixed with ice-cold ethanol, incubated with a blocking solution containing 5% normal goat serum (catalog S-1000; Vector Laboratories) and 0.1% Triton X-100 (MilliporeSigma), and washed twice with phosphate-buffered saline (PBS). Hepatocytes were stained with rabbit anti–mouse MCT1 Ab (catalog 20139-1-AP; Proteintech) or mouse monoclonal anti–sodium/potassium ATPase Ab (catalog Ab7671; Abcam) and diluted in PBS containing 2.5% normal goat serum, followed by secondary hybridization with Alexa Fluor 488–conjugated goat anti–mouse IgG Ab (catalog A32723; Thermo Fisher Scientific) or Alexa Fluor 555–conjugated goat anti–rabbit IgG Ab (catalog A32732; Thermo Fisher Scientific), respectively. Cells then were incubated with DAPI solution (catalog 340-07971; Dojindo Laboratories) and embedded with FluorSave reagent (catalog 345789; MilliporeSigma). Images were acquired using a TiEA1R microscope (Nikon, Inc., Tokyo, Japan) equipped with a Plan Apo λ ×100 numerical aperture 1.45 oil immersion objective lens (Nikon, Inc.) and a GaAsP detector (Nikon, Inc.) in a fixed image acquisition setting. The fluorescence intensities of the resulting images were analyzed with NIS-Elements.

Paraformaldehyde fixed paraffin-embedded tissue sections were rehydrated and subjected to antigen retrieval in a pressure chamber (2100 Retriever, Aptum Biologics Ltd., Southampton, UK). Unspecific binding was blocked using serum-free protein block (DAKO, Glostrup, Denmark) supplemented with 5% normal donkey serum (D9663, Sigma-Aldrich, Saint Louis, Mo, USA). Incubation with goat anti-human AIRE antibody and/or mouse anti-human podoplanin for 60 min in RT followed by secondary Alexa Fluor labeled antibodies (Table S2). Thresholds of positive signal in the confocal microscopy were set using normal goat serum (S-1000, Vector Laboratories, Burlingame, CA, USA) or normal rabbit serum (DAKO, Glostrup, Denmark) followed by the secondary antibody.