Anti iba1 polyclonal antibody

We performed immunohistochemistry at P 17 (n = 4) and P 28 (n = 4). Under deep anesthesia with pentobarbital (>50 mg/kg), rats were perfused with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS). The brains were obtained, post-fixed with the same fixative overnight, and cryoprotected with 30% sucrose. Coronal-sections (40 μm) were prepared from 1–3 mm posterior to the bregma that contains the sensorimotor cortex of the hindlimb area. After soaking with 0.25% Triton X-100 in PBS (PBS-T), blocking was performed with 10% normal goat serum (NGS) (Vector Labs, USA) for 60 min. The slices were reacted with anti-Iba1 polyclonal antibody (1:1,000; Wako, Japan) immersed in PBS-T containing 1% NGS at 4°C overnight, followed by immersion goat anti-rabbit IgG conjugated with Alexa Fluor 594 (1:1,000; Abcam, UK). After the slices were embedded on a glass slide with mounting medium (Vector Labs, USA), slices were photographed with a fluorescence microscope (Axio Observer.Z1; Zeiss, Germany) and an AX70 microscope (Olympus, Japan).

Mouse brains were fixed in 10% buffered formal saline (BFS) and prion-infected tissue was treated in 98% formic acid for one hour to remove infectivity. Tissues were paraffin wax embedded, sagitally sectioned and stained as previously described [22 (link)]. Haematoxylin and eosin (H&E) stained sections were assessed to determine the relative levels and distribution of spongiosis and neuronal loss. Prion deposition was evaluated with the anti-PrP monoclonal antibody ICSM35 (D-Gen Ltd, UK) and gliosis was determined with an anti-glial fibrillary acid protein (GFAP) antibody (Dako Ltd, UK). Microglia were visualised by staining with an anti-Iba1 polyclonal antibody (Wako).

Rats were sacrificed after 24 h of aSAH induction by injection of pentobarbital (200 mg/Kg, i.p.) and perfused trans-cardially with 50 mL of saline followed by 500 mL of a fixative containing 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.3 for 30 min. Serial coronal brain sections of brain cortex, 6 mm thick, were cut on a cryostat, thaw-mounted onto gelatin-coated slides, and were used for immunohistochemical staining.
The samples were de-paraffinized by heating at 60 °C for 30 min and xylene. They were then rehydrated by passing through a series of decreasing concentrations of ethanol (100%, 90%, 70%, and 50%) for 5 min each step, and then washed with 0.1 M phosphate-buffered saline. Endogenous peroxidase was quenched with 3% hydrogen peroxidase for 10 min. The sections were incubated with 5% Bovine serum albumin for 1 h to block nonspecific background staining. Subsequently, they were incubated with primary antibody overnight at 4 °C and visualized using the Novolink Polymer Detection System. Iba-1-positive cells were used as a direct indicator of neuroinflammation. The following immunoreagents were used in this study: Anti-Iba-1 Polyclonal Antibody (019–19741; Wako), Novolink Polymer Detection System (RE7140-K; Novocastra, Newcastle upon Tyne, UK).