Anti iba1 antibody

Paraffin sections were dewaxed, and rehydrated using xylene/ethanol baths and then heated at 95 °C in a 10 mM/pH 6.0 sodium citrate bath for 10 min. The detection of activated HSCs was performed using anti α-SMA antibody (a gift from Christine Chaponnier, Geneva University) (1:50). The detection of macrophages was performed using an anti IBA-1 antibody (Wako Chemicals, Richmond, VA, USA) (1:500). The liver sections were incubated overnight at 4 °C with primary antibody diluted in PBS containing 0.1% BSA, washed in PBS, and incubated for 1 h with a secondary antibody (Alexa Fluor 488 goat-anti mouse) diluted (1:1000) in PBS containing 0.1% BSA. All sections were stained with 0.09% Evans blue solution. Images of immunostained sections were acquired using Axiophot microscope and Axiocam color camera. The percentage of positive cells for α-SMA or IBA-1 was determined using MetaMorph, Image J and Definiens Software. Cells area positive for α-SMA or IBA-1 staining were counted and normalized to the total liver surface. An average of 3000 cells were counted on 4 histology fields per animal.

After the treatments of Ramelteon and LPS or MPTP described above, the mice were perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The mice brains were then removed and post‐fixed in the same fixation agent overnight at 4°C, followed by a treatment with 30% sucrose at 4°C with another night. Serial 20 μM‐thick mouse midbrain slices were cut with a freezing microtome. Immunohistochemical staining was conducted with anti‐MT1 antibody, anti‐Iba1antibody (Wako Chemicals), anti‐GFAP, and anti‐TH antibodies (Millipore) against six slices per mouse (120 μm interval). After incubation with primary antibodies at room temperature for 6 hr, the slices were incubated with rhodamine (red)‐or FITC (green)‐conjugated secondary antibody (Invitrogen) for 2 hr. Thereafter, the slices were stained with DAPI for 5 min and observed using an inverted IX71 microscope system (Olympus). The number of TH⁺ neurons and the fluorescence intensity of MT1, Iba1, and GFAP were counted using Image J (National Institute of Health).

C57BL/6 mice were pretreated with RRx-001 to establish an in vivo model of neuroinflammation. Seven days after LPS injection, the mice were perfused with precooled PBS and then 4% paraformaldehyde and their brains were taken and immersed in 4% paraformaldehyde for 3 days at 4°C. The brains were then dehydrated at 4°C for 3 days in 20 and 30% sucrose, respectively. After being frozen overnight at −80°C, 20 µM-thick slices of the midbrain were obtained at −20°C by using a CM1950 Freezing Microtome (Leica, Wetzlar, Germany). Microglia and astrocytes were visualized by immunohistochemical staining with anti-Iba1 antibody (Wako Chemicals, Tokyo, Japan) and anti-GFAP antibody (Sigma-Aldrich, St. Louis, United States), respectively. The dopaminergic neurons were shown by immunohistochemical staining with anti-tyrosine hydroxylase (TH) antibody (Millipore, Billerica, United States). The nuclei were stained with DAPI. Finally, the slices were imaged by using an inverted Nikon Eclipse Microscope (Nikon, Tokyo, Japan) and the fluorescence intensity was analyzed by using ImageJ software.

The primary antibodies included anti-IBA1 antibody (1:100, Wako), anti-CD68 antibody (1: 100, Abcam, Cambridge, MA, United States), anti-CD16/32 antibody (1:100, BD Biosciences), anti-CD206 antibody (1:100, R&D Systems, Inc., Minneapolis, MN, United States), anti-CD11b antibody (1: 100, Abcam, Cambridge, MA, United States), anti-CD86 antibody (1: 100, Abcam, Cambridge, MA, United States), anti-CD40 antibody (1:100, Abcam, Cambridge, MA, United States), anti-CD163 antibody (1:100, Santa Cruz, MA, United States), anti-TMEM119 antibody (1: 100, Abcam, Cambridge, MA, United States), anti-Rhodopsin antibody (1:100, Santa Cruz, MA, United States). These primary antibodies and abbreviates were explained in Supplementary Table S1, including the description of the characteristics of M1 and M2 markers. Secondary antibodies included donkey anti-rabbit IgG (H+L) Alexa Fluor^® 555, goat anti-rat IgG (H+L) Alexa Fluor^® 488, donkey anti-rabbit IgG (H+L) Alexa Fluor^® 488, and donkey anti-goat IgG (H+L) Alexa Fluor^® 555 secondary antibodies (1:800, Invitrogen).

Isolated CD11b⁺ cells from brain or BM were fixed and permeabilized using fixation/permeabilization solution (BD Biosciences, San Jose, CA, USA) for 20 min at 4°C. After washing, the cells were incubated with anti-Iba1 antibody (Wako) for 30 min at 4°C followed by incubation with Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific) for 30 min at 4°C. The median of fluorescent intensity (MFI) was measured in total 5,000-count cells using a Beckman Coulter Gallios flow cytometer and Kaluza for Gallios software version 1.3 (Beckman Coulter, Indianapolis, IN, USA).

Mice were perfused with ice-cold phosphate-buffered saline (PBS). Brain tissues were separately used for either molecular analysis or immunohistochemistry. The right hemisphere was fixed with 4% PFA and was transferred to a sucrose solution (30%) for 24 h. The right hemisphere was embedded and frozen in optimal cutting temperature compound on dry ice. Coronal Sections (18 μm thick) that included the hippocampus were cut and prepared on slide glasses. Tissue sections were then permeabilized and blocked using a blocking solution (5% goat serum and 0.2% Triton X-100 in PBS) for 1 h at room temperature. Primary antibody incubation was performed overnight at 4 °C using an anti-6e10 antibody (Biolegend, Japan, 1:100), anti-Iba1 antibody (Wako, Japan, 1:200), and anti-GFAP antibody (Abcam, Cambridge, UK, 1:200). After the washes, sections were incubated with the appropriate secondary antibody (1:500) for 1 h at room temperature. Slides were counterstained in Hoechst and then mounted in fluorescence mounting medium (Dako, Denmark). All images were taken through an Axioplan2 microscope (Zeiss, Germany). Fluorescence was measured by calculating % area and was analyzed using ImageJ. Sample sizes were 10–15 mice per group.