Ab75476

FFPE sections were rehydrated through graded alcohol and epitopes were retrieved by overnight incubation in sodium citrate buffer at 60 °C. Sections were quenched with 3% hydrogen peroxide in PBS for 15 min at room temperature then blocked for 1 h with 5% BSA in TBST at room temperature. Sections were probed overnight with rabbit polyclonal to F4/80 (1:200 ab75476; Abcam, Cambridge, MA, USA) followed by secondary goat-anti-rabbit HRP-conjugated antibody (ab97051, Abcam) at a dilution of 1:500. Immunoreactivity was revealed using ImmPACT DAB (Vector Labs, Burlingame, CA, USA) and sections were counterstained with hematoxylin, dehydrated and mounted with Cytoseal XYL (Thermo Scientific, Waltham, MA, USA). Positively stained cells were counted in three high-power field areas (100× oil immersion) in each liver cross section.

Tissues were fixed in 10% neutral-buffered formalin. Tissue processing, paraffin embedding, sectioning, and immunohistochemistry staining were performed by the MD Anderson Department of Veterinary Medicine and Surgery Histology Laboratory. Immunohistochemistry was performed using standard methods, and tissue sections were stained with antibodies against CD11b (ab75476, Abcam). Tissue sections were imaged on a Zeiss Axioplan Imaging 2e infinity-corrected upright microscope equipped with Color Zeiss Axiocam.

Paraffin-embedded (5μm) tissue sections were pre-treated for deparaffinization, rehydration and epitope retrieval using an automated PT-link system (Dako) and stained with polyclonal rabbit antibodies against: GRIA2 (Abcam ab52176, 1:200), CD11b (Abcam ab75476, 1:600) or monoclonal mouse antibody against Ki67 (Dako M7240, 1:100) for 30min at room temperature. The staining was visualized by using DakoCytomation EnVision+ System-HRP suitable for rabbit or mouse primary antibodies. Sections stained with polyclonal rabbit/mouse IgG followed by the EnVision+ System was used as negative controls.

The hippocampus samples were homogenized on ice in cold radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF) protease inhibitor, and the lysates were centrifuged at 4°C, and 12,000 rpm for 5 min. The BCA protein assay kit (Beyotime Biotechnology, Nantong, Jiangsu, China) was used to determine the total protein concentration of each sample. The proteins of each sample were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. Subsequently, the membranes were blocked with 5% skim milk dissolved in Tris-buffered saline for 1 h and incubated with primary antibodies overnight. The primary antibodies included antibodies against BDNF (1:1,000, ab108319, Abcam, Cambridge, UK), P75 (1:1,000, ab52987, Abcam, Cambridge, UK), TrK B (1:1,000, ab18987, Abcam, Cambridge, UK), CD11b (1:1000, ab75476, Abcam, Cambridge, UK), GFAP (1:2,000, sc-33673, Santa Cruz, CA, USA), GDNF (1:1,000, ab176564, Abcam, Cambridge, UK), and β-actin (1:2,000, sc-47778, Santa Cruz, CA, USA). Then, the membranes were washed with TBST three times and incubated with an HRP-labeled secondary antibody for 1 h at room temperature. The bands were detected with an enhanced chemiluminescence (ECL) system and quantified by Image J Software.

The mouse femur with a cavity created by K‐files was fixed in 4% formaldehyde for 48 hrs at 4°C and washed with running tap water for 5 hrs. Decalcification was performed with 10% EDTA by exchanging the solution after every 2–4 days until the tissue softened. The samples were dehydrated with ascending concentrations of ethanol, followed by clearing with xylene before embedding in paraffin (histoparaffin m.p. 56–58°C; Wako Pure Chemical). Five micrometre‐thick paraffin sections were made, de‐paraffinized and processed for immunohistochemistry. The section was blocked in G‐Block (Genostaff GB‐01; Genostaff Co., Ltd, Tokyo, Japan) at room temperature and incubated at 4°C overnight with primary antibodies, F4/80 (0.4 μg/ml) (ab6640 abcam^®) and CD11b (1 μg/ml) (ab75476 abcam^®), for macrophages and monocytes, respectively. The secondary antibodies were anti‐rabbit biotin (Dako E0432) and anti‐rat biotin (Dako E0468) for monocytes and macrophages, respectively. The sections were further reacted with streptavidin (Nichirei Biosciences 426062, Tokyo, Japan) for 5 min. at room temperature followed by DAB/H₂O₂ and haematoxylin and eosin (H & E) staining. The sections were sealed with Malinol 750cps^® (Muto Pure Chemicals, Tokyo, Japan) for observation.