Biotinylated goat anti rabbit

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.

We studied the colocalization of [³H]-thymidine-labeled cells with DCX or GFAP in the experimental groups with survival times of 1.5 h and 3 days after administration of [³H]-thymidine. For this purpose, we obtained four series of semithin sections of three telencephalic levels: pre-commissural telencephalon, anterior olfactory nucleus (AON), and olfactory peduncle (sectioned longitudinally). In the first and third series we performed autoradiographic detections of [³H]-thymidine labeled cells. In the second and fourth series we performed immunohistochemistry detection for DCX and GFAP, respectively. Post-embedding immunohistochemistry was performed as previously described (Crespo et al., 2003 (link)). The primary antibodies used were polyclonal rabbit anti-glial fibrillary acidic protein (1:500, Dako) or polyclonal goat anti-doublecortin (1:200, Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibodies were biotinylated goat anti-rabbit and biotinylated rabbit anti-goat (Vector laboratories), respectively. We then performed studies correlating DCX- and GFAP-positive cells with the analysis of their ultrastructure in adjacent semithin sections in which they were co-labeled with [³H]-thymidine.

Slides of cultured human ovary were examined to identify sections that did not contain ovarian follicles, with reactions carried to determine rates of proliferation through IHC for BrdU incorporation (rat anti-BrdU antibody, Abcam, dilution 1:200) and of apoptosis through IHC for cleaved caspase 3 (CC3; rabbit anti-CC3 antibody, Cell Signalling Technology, dilution 1:500); n = 11 for both. Washes in PBS (Fisher Scientific UK Ltd) with 0.1% Triton X (PBSTx) were performed between each step. Antigen retrieval was performed in 0.01 M citrate buffer (pH 6, Sigma Aldrich Ltd), followed by blocking step with 20% normal goat serum diluted in PBSTx and 5% w/v BSA for 1 h at RT. Slides were incubated with primary antibodies overnight at 4°C in a humidified environment followed by incubation with appropriate secondary antibody and visualisation reagent all at 1:200 dilution: for BrdU IHC, AlexaFluor 568 nm goat anti-rat (Invitrogen) was used; for CC3 IHC, goat anti-rabbit biotinylated (Vector Laboratories), was followed by Streptavidin 488 (Vector Laboratories). Counterstaining was with 4,6-diamidino-2-phenylindole (DAPI; Invitrogen) at 1:5000 for 10 min and slides were then mounted with Vectashield hard-set mounting medium (Vector Laboratories).