Rabbit anti iba1

To evaluate the ultrastructural characteristics of DAI in the micro pig thalamus while verifying microglia process contacts on these axonal swellings, a subset of tissue was immunolabeled with either rabbit anti β-APP (1:700; Cat.# 51–2700, Life Technologies, Carlsbad, CA, USA) or rabbit anti Iba-1 (1:1000; Cat.# 019–19741, Wako, Osaka, Japan), followed by incubation with biotinylated goat anti-rabbit IgG (1:1000; Cat.# BA-1000, Vector Laboratories, Burlingame, CA, USA) secondary antibody. The reaction product was visualized with 0.05 % diaminobenzidine/0.01 % hydrogen peroxide/0.3 % imidazole in 0.1 M phosphate buffer and the tissue was prepared for EM analysis. In this approach, tissue sections were osmicated, dehydrated, and embedded in epoxy resin on plastic slides. After resin curing, the slides were studied with routine light microscopy to identify the precise thalamic areas for excision. Once identified, these sites were removed, mounted on plastic studs, and 70-nm sections were cut serially and mounted on Formvar-coated slotted grids. The grids were stained in 5 % uranyl acetate in 50 % methanol and 0.5 % lead citrate. Ultrastructural qualitative analysis was performed using a JEOL JEM 1230 transmission electron microscope (JEOL-USA, Peabody, MA, USA) equipped with Ultrascan 4000SP CCD and Orius SC1000 CCD cameras (Gatan, Pleasanton, CA, USA).

For immunohistochemistry, the following primary antibodies were used: rabbit anti-Iba1 (1:1,000, Wako, Japan) and mouse anti-CD68/ED1 (1:200, AbD Serotec, Germany). Sections were incubated with an appropriate primary antibody overnight at 4°C in blocking serum. This was followed by 2 h of incubation in secondary antibody at room temperature. For each immunohistochemical assessment, some brain sections went through the entire protocol without primary antibody incubation, to serve as the negative controls. The following secondary antibodies were used: Cy3-conjugated donkey anti-mouse/ rabbit (Jackson Immunoresearch, UK), or Alexa-488 conjugated streptavidin (1:200, Invitrogen, Sweden). The sections were mounted on gelatin-coated slides and coverslipped using a glycerol-based mounting medium (DABCO, Sigma).

Primary neurons and N9 microglial cells were washed and fixed with 4% paraformaldehyde (PFA) in PBS. Cells were permeabilized with 0.25% Triton in PBS prior to blocking and overnight incubation at 4 °C with primary antibodies: mouse anti-β3-Tubulin (Biolegend) and rabbit anti-Iba1 (Wako) for neurons and microglia, respectively. Secondary antibodies anti-mouse Alexa 488 (Cell Signaling Technologies) and anti-rabbit Alexa 594 (Invitrogen) were incubated for 1 h at RT. Nuclear staining was performed by incubating cells with Hoechst (Sigma) for 5 min at RT. Coverslips were mounted in microscope slides with Fluoroshield (Sigma) and images randomly acquired in a Zeiss Axio Imager Z1 Apotome. Neuronal apoptosis was addressed by evaluating nuclei shape of ten images per condition²⁷ .

Frozen brain tissues were blocked with 10% goat serum in 0.01 M PBS for 1 h and then incubated overnight at 4 °C with primary antibodies (Abs). Brain sections (20 μm) were incubated with the following primary Abs: mouse anti-KCa3.1 (1:100; Alomone Labs, Ltd., Jerusalem, Israel) and rabbit anti-Iba1 (1:500; Wako Pure Chemical Industries, Ltd., Osaka, Japan). The brain sections were then washed with 0.01 M PBS and incubated with the respective Alexa Fluor® 488- or 568-conjugated secondary Abs (1:500; Invitrogen Corporation). Fluorescent images were acquired using a TCS SP8 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). For imaging acquisition, a prescan of all samples was performed to ensure confocal settings below saturation. For each experiment, all images were obtained using the same confocal settings. Six slices at 120 μm intervals from each brain were used to examine Iba1-positive cells. Three microscopic fields (0.01 mm²) were randomly selected in each slice with the same reference position for quantification. The Iba1-positive cell number was counted in a blinded manner, and the area was measured by Leica LAS AF Lite software (Leica, Germany).