Anti gfap antibody

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.

Immunohistochemistry experiments were performed on free-floating brain sections (25 μm) from vehicle, and RGFP966-treated N171-82Q transgenic mice, as described previously [22 (link)], using an anti-GFAP antibody (Abcam; 1:500 dilution) or anti-Huntingtin, EM-48 (Abcam; 1:500 dilution). The immunoreaction was detected with Vectastain ABC kit (Vector Laboratory Inc., Burlingame, CA) according to the instructions of the manufacturer. Enzymatic development was performed in 0.05% diaminobenzene in PBS containing 0.003% hydrogen peroxide for 3–5 min. Quantification of GFAP-positive immunoreactivity was performed by counting the number of labeled cells per microscopic field using a 63x oil objective. For each brain, values from n = 5–10 striata were averaged to obtain a single value per mouse. Significant differences due to RGFP966 treatment were determined using Student’s t test (unpaired; two-tailed) (GraphPad Prism, San Diego, CA).

Animals were euthanized with CO₂ at 4, 8, and 12 weeks postoperation, after which eyeballs were enucleated and retinas were quickly isolated on ice. After being rinsed in 0.01 M PBS and drained, retina tissues were lysed in ice-cold tissue lysis buffer [10% phenylmethylsulfonyl fluoride (PMSF) + 90% radioimmunoprecipitation assay (RIPA)]. The lysates were then centrifuged at 15,000 rpm for 10 min at 4°C. Protein concentration was determined using the BCA Protein Assay (Beyotime). After boiling in loading buffer for 10 min, total proteins (10 μg per slot) were electrophoresed on a 12% sodium dodecyl sulfate polyacrylamide gel and then transferred onto polyvinylidene fluoride membranes. After being blocked in 5% fat-free milk for 2 h at 37°C, membranes were incubated with anti-GFAP antibody (1:500, rabbit, Abcam) and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (1:1,000, mouse, Proteintech Group) antibody overnight at 4°C. Membranes were then incubated with peroxidase-conjugated immunoglobulin G (1:2,000; Santa Cruz Biotechnology). After being washed in Tris-buffered saline with Tween-20 (TBS-T) and developed in developing solution, membranes were scanned using the Bio-Rad exploding system (Bio-Rad, CA, United States) with corresponding software.

1,4-Dideoxy-1,4-imino-D-arabinitol (DAB), L-lactate, isofagomine, and internal control antibody anti-α-Tubulin mouse mAb (DM1A,1:5000) were purchased from Sigma (St. Louis, MO, U.S.A.). Primary antibodies of phosphorylated glycogen synthase kinase 3β (GSK3β) at Ser-9 (pS9, 1:1000) and phosphorylated Akt at Ser-473 (p-Akt, 1:1000) were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Antibodies used to identify axons and dendrites, tau-1 (1:200), and antimicrotubule-associated protein-2 (MAP-2, 1:200) were purchased from Millipore (Billerica, MA, U.S.A.). Anti-GFAP antibody was purchased from Abcam (Cambridge, UK). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Japan. Neurobasal and B27 were purchased from Invitrogen (Grand Island, NY, U.S.A.)

Bone marrow-derived macrophages and primary mouse astrocyte cultures were prepared, as previously described [28 (link)]. Briefly, femur and tibia were removed from euthanized C57BL/6 mice under sterile conditions. The two ends of bones were cut, and bone marrow was expelled with a syringe filled with culture medium. Cells from bone marrow were cultured for 7 days in the presence of 50 ng/ml recombinant macrophage colony-stimulating factor (M-CSF) (eBioscience) [28 (link)]. Differentiated macrophages were treated with lipopolysaccharide (LPS) (10 and 100 ng/ml) for 12 h at 37 °C before RNA extraction. For astrocyte cultures, neonatal mouse brain tissue was used. Brains were removed and placed in DMEM medium under sterile conditions. Brain tissues were dissected, and astrocyte cells were cultured in DMEM medium supplemented with 20% FBS. Astrocytes were stimulated with 10 and 100 ng/ml LPS (Sigma Aldrich) for 12 h at 37 °C [29 ]. To confirm the identity of the cells, we performed immunofluorescent staining using an anti-GFAP antibody (1:250, mouse polyclonal, Abcam).