Biotinylated goat anti guinea pig

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.

For the detection of migrating immature neurons or neuroblasts within the SVZ sections were fixed in 4% paraformaldehyde (PFA in 0.1 M PBS, pH 7.4, for 15 min RT) and then washed in PBS (0.1 M; 3×5 min) prior to antigen retrieval with 10% proteinase K (DAKO, Glostrup, Denmark) in 0.1 M PBS for 3 min following by incubation in 30% fetal calf serum to stop proteinase K activity for 30 min. Sections were washed in PBS (0.1 M; 3×5 min) followed by incubation in blocking solution containing 5% NGS in 0.1 M PBS for 30 min. Sections were then incubated with guinea-pig anti-doublecortin (DCX; 1∶3000, Millipore, Billerica, MA, USA) for 1 hour in a mixture of PBS (0.1 M; pH 7.4) containing 5% NGS prior to PBS washes (0.1 M; 3×5 min) and incubated with secondary antibody biotinylated goat anti-guinea-pig (1∶200, Vector Labs, Burlingame, CA, USA) for 30 min. Sections were further analysed as described above.