Rabbit anti iba 1 polyclonal antibody

Immunostaining was performed as we previously reported [47 (link), 48 (link)]. In brief, fixed coronal brain section (25 µm) underwent 60-min permeabilization and blocking with PBS containing 0.2% Triton X-100, 10% goat serum, and 22.52 mg/mL glycine. The sections were then incubated in rabbit anti-Iba1 polyclonal antibody (Wako cat. #019-19741, 1:200) and rat anti-Siglec-E monoclonal antibody (BioLegend cat. #677102, 1:100) at 4 °C overnight. Slices were then rinsed and incubated with anti-rabbit and anti-rat secondary antibodies conjugated with Alexa Fluor 546 or 488 (1:1000, Invitrogen) for 2 h. The slices were carefully mounted onto slides using the ProLong™ Gold antifade reagent (Invitrogen). The digital images were captured using a fluorescence microscope BZ-X800 (Keyence). The image processing and quantitative analyses were performed using the ImageJ/Fiji software (NIH).

IHC was performed as described by Ma et al. [49 (link)]. The following antibodies were used: mouse anti-NeuN monoclonal antibody (1:1,000, Chemicon, EMD Millipore, Billerica, MA, USA), rabbit anti-GFAP polyclonal antibody (1:1,000, Dako, Glostrup, Denmark), rabbit anti-Iba1 polyclonal antibody (1:500, Wako, Osaka, Japan). Secondary antibodies were labeled with biotin (1:200, Vector Laboratory, Burlingame, CA, USA). After IHC reaction, images were captured using the Nikon digital camera system (DS-Fi1) in combination with microscopy (Nikon Eclipse 80i).

The mice were anesthetized with an overdose of pentobarbital (40 mg/kg), and the heart was perfused with 0.1 mol/L PBS (pH = 7.4) containing 4% paraformaldehyde. The brain was cut into 5-μm-thick coronal sections for immunohistochemical staining. In brief, the sections were incubated with the primary antibodies overnight at 4°C, including rabbit anti-myeloperoxidase (MPO) monoclonal antibody (neutrophils marker, 1:800, Abcam, Cambridge, MA, United States), and rabbit anti-Iba1 polyclonal antibody (microglia marker, 1:200, Wako, Japan). The sections were washed with PBS (pH = 7.4) 3 times, 3 min each time. Sections were then incubated with horseradish peroxidase (HRP) combined with the secondary antibody rabbit immunoglobulin G (IgG) antibody (Servicebio, Wuhan, China) for 1 h at room temperature.

Histological analysis was performed 1 day after the administration of 300 mM 3-NP (n = 3). For tissue fixation, rats were deeply anesthetized with pentobarbital (as described above) and transcardially perfused with 4% paraformaldehyde. Paraffin sections were prepared as previously described [20] (link). Immunohistochemistry for Iba-1 and IL-6 was performed as previously described [6] (link). All slides were washed with PBS, incubated in 1.5% hydrogen peroxide for 15 min, rinsed three times in PBS, incubated with 10% normal goat serum for 1 h at room temperature, and then incubated overnight with the appropriate primary antibodies at 48°C. The primary antibodies used were a rabbit anti-IL-6 polyclonal antibody (diluted 1∶150; Sigma) and a rabbit anti-Iba-1 polyclonal antibody (diluted 1∶500; Wako Pure Chemicals). The slides were then incubated with biotinylated secondary antibodies (1∶1000) at 37°C for 30 min, washed three times in PBS, and then incubated in VECTASTAIN Elite ABC reagent (Elite ABC kit, Vector Laboratories, Burlingame, CA) for 30 min at room temperature. After gentle washing in PBS (three times), staining was visualized by incubating the samples with diaminobenzidine solution (Wako Pure Chemical Industries). After a final wash with PBS, the samples were dehydrated and mounted under coverslips.

Fixed frozen sections of spinal cord from mSOD1^G93A mice (10 μm thick) were prepared as previously described^{32 (link)}. Hypoxia-induced factor 1-alpha (HIF-1α) expression was evaluated in paraffin-embedded 6-μm–thick sections of the lumbar cord from ALS patients and 10 μm–thick fixed frozen sections of spinal cord of mSOD1^G93A mice as previously described^{41 (link)}. The following primary antibodies were used: rabbit anti–HIF-1α polyclonal antibody (1:100; Novus Biologicals), rabbit anti–Iba1 polyclonal antibody (1:1000, Wako) and Alexa Fluor 488®–conjugated mouse anti–glial fibrillary acidic protein (GFAP) monoclonal antibody (1:200; Cell Signaling Technology). The following secondary antibodies were used: AlexaFlour488-conjugated goat anti–rabbit IgG antibody (1:500, Invitrogen) and goat anti–rabbit immunoglobulins conjugated to peroxidase-labeled dextran polymer (Dako Envision+, Dako). For HIF-1α staining, reaction products were visualized with 3,3′-diaminobenzidine tetrahydrochloride (Vector Laboratories). The relative optical densities of HIF-1α immunoreactivity were quantified using ImageJ. The fluorescently labeled sections were examined using an LSM 510 confocal microscope (Zeiss).

Paraffin-embedded, 2-μm coronal brain sections were used to visualize microglia by staining of ionized calcium-binding adapter molecule 1 (Iba-1) with the rabbit anti-Iba-1 polyclonal antibody (Wako Chemicals) (33 (link)). The number of Iba-1⁺ cells was determined in six different neocortical regions and the hippocampal fissure and then divided by the number of scored regions by a blinded observer. The Iba-1 staining revealed four divergent cell morphologies according to gradual steps of microglial activation (38 (link), 39 (link)). Based on the most abundant morphology at a magnification of 20, a microglia activation score (AS) was given to each scored brain region in a blinded way. Microglial activation is a multi-step process that starts with hyper-ramification and subsequent enlargement of the cell body and more pronounced thickness and gradual retraction of the ramifications until acquisition of an ameboid morphology (38 (link)). Microglia with relative big somata but fine ramifications were scored as an AS of 1 (40 (link)). An AS of 2 was given to hypertrophic cells with thicker branches, while AS3 and AS4 were assigned to bushy and ameboid cells, respectively (39 (link)).

Mice were deeply anesthetized with a ketamine/xylazine cocktail and then perfused transcardially with 0.9% saline followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). The lumbar spinal cords were removed and placed in the same fixative overnight, then transferred to 30% sucrose for cryoprotection. Frozen spinal cords were cut transversely into 25 μm-thick sections on a sliding microtome, collected in an anti-freeze solution [0.05 M sodium phosphate buffer (pH 7.3) containing 30% ethylene glycol and 20% glycerol] and stored at −20 °C until use. Standard fluorescent immunohistochemistry protocols were applied with the use of antibodies and dye against selective proteins. Floating sections were washed in TBS 3 times, blocked and incubated for 24 h at 4 °C with primary antibodies: rabbit anti-Iba-1 polyclonal antibody (ionizing calcium-binding adaptor molecule, for microglia and macrophages, 1:1000; Wako, Richmond, VA), CD16/32 (Fcγ receptors III/II) (1:400, R&D Systems, Minneapolis, MN), iNOS (1:200, BD Bioscience). Sections were then incubated for 60 min at room temperature with a corresponding secondary antibody, then counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) for nuclear labeling. After rinses in TBS, sections were mounted onto slides and coverslipped with Vectashield Mounting medium (Vector Lab, Burlingame, CA).