Ba 7000

Immunolabeling for Fos was performed first then followed by kisspeptin. Briefly, sections were incubated successively in 0.1% NaBH₄ (15 min), 3% H₂O₂ (30 min), 10% normal goat serum (1 h) and then 48 h at 4 °C in a guinea pig polyclonal anti-c-Fos antibody (1:1000; Synaptic systems, 226005). Then they were incubated for 1 h in a goat anti-guinea pig biotinylated antibody (1:1000; Vector Labs, BA-7000) followed by ABC complex (1:800; Vector Labs, PK-6100) for 1 h. The peroxidase was visualized with 3,3′-diaminobenzidine to give a brown precipitate. Then, the residual peroxidase activity was blocked in H₂O₂ (3%), followed by a 1-h incubation in 5% normal goat serum. Sections were further processed for kisspeptin using a rabbit polyclonal anti-kisspeptin 10 antibody (1:10000; Alain Caraty, INRA, France) for 48 h at 4 °C. Brains sections were then incubated in a goat anti-rabbit biotinylated antibody (Jackson immuneResearch, 111-065-003) followed by the ABC complex for 1 h, and the peroxidase was visualized by a modified DAB to give a blue precipitate (Vector Labs, SK-4700). Sections were mounted, cleared with xylene and cover slipped with Eukitt mounting medium.

Four weeks after pMCAO, NS and S pigs were euthanized by IV pentobarbital (1 mL/4.5 kg) injection. Brains from both groups were removed and immersed in 10% buffered formalin (Millipore Sigma). Next, brains were sectioned coronally using a pig brain slicer (Zivic Instruments, Pittsburgh, PA). Right (ipsilateral to pMCAO) hemisphere sections were formalin-fixed, embedded in paraffin, and sectioned for immunohistochemistry (Leica RM2255, Germany). Following an enzyme block in 3% H₂O₂ for 5 min and Power Block (BioGenex) for 5 min, 4 μM thick sections were incubated with primary antibodies for 1 h on a Biocare Medical Nemesis 7200 Autostainer. Primary antibodies used were GFAP (mouse 1:4,000, Biogenex MU020-UC, Clone GA-5), IBA1 (rabbit 1:8,000, Wako, 019-19741), NeuN (guinea pig 1:3,000, Millipore Sigma, ABN90), FactorVIII (rabbit, Cell Marque, 250A-18), and DCX (rabbit, 1:4,000, Abcam, ab18723). Secondary antibodies used were Biotinylated goat anti-rabbit 1:100, Vector Labs, BA-1000 for IBA1, Biotinylated horse anti-mouse 1:100, Vector Labs, BA-2001 for GFAP, Biotinylated goat anti-guinea pig 1:100, Vector Labs, BA-7000 for NeuN, Biotinylated goat anti-rabbit 1:100, Vector Labs, BA-1000 for FactorVIII, and Biotinylated goat anti-rabbit 1:100, Vector Labs, BA-1000 for DCX. The substrate is DAB + Chromogen (12 min) from Biocare and all were counterstained with hematoxylin.

Briefly, 96-well plates (Nunc, 96 F MAXISORP, Roskilde, Denmark) where coated with formalin-inactivated Influenza A virus H1N1 (SBL Influenza Vaccine, Sanoil Pasteur, Lyon, France) diluted in coating buffer (0.05 M sodium carbonate buffer, pH 9.5–9.7) at 5 μg/mL and incubated at +4 °C over night. Wells were washed x3 (0.9% NaCl and 0.05% Tween-20) and blocked with 3% BSA in PBS buffer for 1 hour at 37 °C. Serum samples were diluted 1:100 and further in two-fold dilutions in dilution buffer (PBS containing 0.5% BSA and 0.05% Tween-20), and incubated for 90 min at 37 °C. Plates were then washed x5 and incubated for 60 min at 37 °C with secondary biotinylated goat-anti guinea pig antibody (Vector, BA-7000) and horseradish-peroxidase (HRP) conjugated Streptavidin (DAKO, Denmark, P0397), both at a dilution of 1:3000. Plates were then washed x5 and 100 μL of tetramethyl benzidine (TMB) substrate (Sigma Aldrich, T-0440-16) was added to each well, the reaction developed for 10 min and stopped by addition of 100 μL of 2 M H₂SO₄. Absorbance was measured at 450 nm in an ELISA reader (VersaMax, Molecular Devices). Cut off values were calculated as the average value of negative controls OD and 2 times the SD.