Pig brain slicer

At weaning (PND 19.97 ± 0.14), all piglets were euthanized via CO₂ asphyxiation. The brains were collected and coronally sectioned using a pig brain slicer (Zivic Instruments, Pittsburgh, PA, USA) and fresh tissues of HC and PFC were collected and immediately frozen in liquid nitrogen and stored in −80 °C for future analysis.

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.