Animals were fully anaesthetized and perfused with saline and 4% formaldehyde. The brain was post-fixed with 4% formaldehyde in PBS for one day at 4 °C. Afterward, the brain was dehydrated with 30% sucrose in PBS for at least 3 days and then it was rapidly frozen with OCT (optimal cutting temperature) compound. Coronal sections (20 μm) were cut using a cryotome. The sectioned tissues were incubated with 3 % bovine serum albumin and 0.3% Tx-100 in PBS for 1 h at room temperature and incubated with the primary antibody (GFAP, monoamine oxidase B (Mao B) and Iba-1; Cell Signaling Technology) overnight at 4 °C. The fluorescence-conjugated secondary antibody (1:200; Jackson) was applied for 1 hour at room temperature. Tissues were mounted with Vectashield with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, USA). The stained sections were observed using laser scanning (LSM 700 confocal) microscopy. For semi-quantitative estimation of the staining intensity of Mao B expression, the fluorescence intensity of Mao B-stained regions was analyzed by calculating their mean fluorescence intensity using Zen 2010 software (Carl Zeiss). Sholl analysis was performed using Image J (FIJI-Image J).
Iba 1
Manufactured by Cell Signaling Technology
Sourced in United States
Iba-1 is a marker protein that is specifically expressed in microglia, the resident macrophages of the central nervous system. It is commonly used in immunohistochemistry and western blotting applications to identify and analyze microglial cells.
Automatically generated - may contain errors
Lab products found in correlation
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (7 mentions)
Neuronal nuclei (neun), by Cell Signaling Technology (6 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Nf κb p65, by Cell Signaling Technology (3 mentions)
Ecl western blotting detection system, by Merck Group (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Bcl 2 associated x factor bax, by Abcam (2 mentions)
Cd206, by Abcam (2 mentions)
Un scan it 6, by Silk Scientific (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
Parvalbumin, by Cell Signaling Technology (2 mentions)
Sp5 2 confocal microscope, by Leica (2 mentions)
Hdac3, by Abcam (2 mentions)
Lamin b1, by Cell Signaling Technology (2 mentions)
Vectashield with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Fluorescence conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Zen 2010, by Zeiss (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
36 protocols using iba 1
1
Neuroinflammation Molecular Profiling by IHC
2
Western Blot Analysis of Cellular Proteins
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Lysates from cells and tissues were subjected to 10 % SDS-PAGE (Bio-Rad, Pre-gel, MA); separated proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Seoul, Korea). Membranes were blocked with TBS-based blocking buffer (Li-COR Biosciences, Lincoln, NE) for 1h at room temperature, and incubated overnight at 4 °C with primary antibodies p16, SOD1,SOD2, ERK, p-ERK, CREB, p-CREB, p-elF2α, Beclin1, Bip, ATG12, LC3B, p62, BNIP3, PINK1, Parkin, PPARα, FATP4, Tau, P-tau, Beta-amyloid, GFAP, iba-1, beta-actin (1:1000, Cell Signaling Technology, Danvers, MA). Membranes were then washed with TBST three times for 10 min each and incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000, Cell Signaling Technology, Danvers, MA) for 1 h at room temperature and washed again. Signals were detected by Odyssey-LC chemiluminsescent imaging system (LI-COR, Lincoln, NE). Signals were averaged and expressed as described in figure legend.
3
Protein Expression Analysis in Retina and Cells
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The retina of mice and BV-2 or HRECs were collected and homogenized with lysis buffer, followed which centrifugation (1600 × g, 15 min) was performed at 4°C. Protein concentration was quantified in supernatant fluid by bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China). Separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to the PVDF membranes (GE Healthcare Europe GmbH, Freiburg, Germany). Proteins were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies (p-p65: ab76302; t-p65: ab32536; occludin: ab216327; claudin-1: ab180158; ZO-1: ab216880; VEGF: ab214424; CD31: ab182981; GAPDH: ab8245. Abcam, England. p-IκB: #2859, t-IκB: #9242, Iba-1: #17,198, Cell Signaling Technology, USA). Subsequently, HRP-conjugated secondary antibody (ab7090, abcam, England) was used to incubate the membranes for 1 h at 37°C. Protein bands were visualized by enhanced chemiluminescence reagent (Millipore Corp., Bedford, MA).
4
Microglia Phenotypic Characterization in Tissue
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Mice were sacrificed and perfused with 50 mL cold PBS, followed by 50 mL 4% paraformaldehyde. Cryosections were obtained of the coronal plain at 5 μm thickness. Non-specific staining was blocked by 1% BSA for 1 h, followed by incubation with primary antibodies against microglia surface marker CD11b (1:100 Cell Signaling, Danvers, MA, USA), M2 marker CD163 (1:120 Cell Signaling) for 2 h at 4 °C. Slides were washed thrice before staining with secondary antibodies: anti-rabbit (Cell Signaling) and anti-mouse (Cell Signaling). DAPI nuclear staining (Sigma-Aldrich) was added to the mounting solution. Primary-cultured microglia were stained as follows: cells were fixed with 4% paraformaldehyde for 1 h, washed thrice with cold PBS, and stained with antibodies against Iba-1(1:100, Cell Signaling) for 2 h with nuclear staining DAPI (Sigma-Aldrich) added at the last 15 min. Cells were washed twice before incubation with secondary antibodies: anti-mouse (Cell Signaling) and anti-rabbit (Cell Signaling). All images were taken by the Invitrogen EVOS FL Color imaging system.
5
Immunohistochemical Analysis of Mouse Brains
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Mice used for immunohistochemical analysis were anesthetized with isoflurane and transcardially perfused with saline followed by 4% paraformaldehyde. Brains were harvested and postfixed in 4% paraformaldehyde overnight at 4 °C. After fixation brains were incubated sequentially in 10%, 20%, and 30% sucrose solutions, each for 24 h at 4 °C. Next, brains were frozen and cut on a cryostat as 30 µm coronal sections. Floating sections were collected and processed for immunohistochemistry and then mounted on glass slides. To aid in visualizing brain structures, nuclei were counterstained with Hoechst dye (0.5 μg/mL final concentration, ThermoFisher Scientific, Waltham, MA, USA). The primary antibodies used include Doublecortin (DCX) (Cell Signaling, cat. No: 4604, host: rabbit, dilution used 1:800), and IBA1 (Cell Signaling, cat. No: 17198S, host: rabbit, dilution used 1:500). The secondary antibody used was donkey-anti-rabbit CY3 (Jackson Immuno Research, cat. No: 711-165-152, dilution used: 1:500). Sections were imaged with the EVOS microscope or ImageXpress Pico instrument using 4×, 10×, or 20× magnification.
6
Protein Expression Analysis in Neuroinflammation
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Proteins were extracted with radioimmunoprecipitation assay lysis buffer (sc-24948; Santa Cruz Biotechnology). Proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane that was probed with primary antibodies against B cell lymphoma (Bcl)-2 (1:400), Bcl-2-associated X factor (Bax) (1:200), CD16 (1:200), CD206 (1:200); and GFAP (1:400) (all from Abcam); and cleaved caspase-3 (1:200), Iba-1 (1:100), and NF-κB p65 (1:200) (all from Cell Signaling Technology), followed by incubation with appropriate secondary antibodies. Immunoreactivity was visualized with the ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). Gray value analysis was conducted with the UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA). Expression levels were normalized against β-actin (1:5000, Boster Biotech) or laminin B1 (1:3000, Cell Signaling Technology).
7
Neuroinflammation Regulation by PDE4B
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Ethanol and lipopolysaccharides (Escherichia coli 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) was purchased from American Type Culture Collection (ATCC). Antibodies GAPDH (Rabbit mAb, cat# 2118S), IBA-1(Rabbit mAb, cat# 17198S), NLRP3 (Rabbit mAb, cat# 15101S) and IL-1β (Mouse mAb, cat# 12242S) were bought from Cell Signaling technology and used at (1:1000) dilution as per the manufacturer’s instructions. PDE4B (Rabbit mAb, cat# ab170939) antibody was obtained from Abcam. Secondary antibodies were obtained from Sigma Aldrich. Fetal bovine serum and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA). Primers were obtained from IDT (Coralville, IA, USA).
8
Western Blot Analysis of Neuroimmune Markers
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Brain tissues were obtained at 28 DPI and total protein was lysed with RIPA lysis buffer (Solarbio), PMSF (Solarbio), and phosphatase inhibitor (Sigma). Equally loaded proteins were electrophoretically separated on 10% and 15% SDS-PAGE gels and then transferred to PVDF membranes (Millipore). After being blocked by silk milk, membranes were incubated with primary antibodies for Phospho-Tau, Iba1, Phospho-p38 MAPK (1:1000, Cell Signaling Technology), and GAPDH (1:1000, Abcam). Horseradish peroxidase-conjugated secondary antibodies were used as a chromogenic reagent (1:5000, Zhongshanxinqiao).
9
Immunofluorescence Analysis of Spinal Cord
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In accordance with the manufacturer’s instructions, the expression of GAP-43, Iba-1, TXNIP, cleaved-Caspase-1 and NLRP3 expressions in four to six lumbar cord segments (L4-L6) were detected by immunofluorescence. Then, the samples were blocked with 1% BSA and 0.1% Triton X-100 for 60 min, GAP-43, Iba-1, TXNIP, cleaved-Caspase-1 and NLRP3 (1:500, Cell signaling, Beverly, MA, USA) antibodies were added. Secondary antibodies IgG (H + L) FITC (1:1000, 11–4011-85, ThermoFisher) were added and incubated in the dark for 2 h. The images were taken and quantified with the A Zeiss LSM 800 confocal laser scanning microscope at a magnification of 20×.
10
Protein Isolation and Western Blot Analysis of Microglia
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Brain samples were isolated from P9 rat pups of both sexes and were homogenized with a lysis buffer containing 150 mmol/L NaCl, 50 mmol/L Tris HCl, 10 mmol/L EDTA, 0.1% Tween 20, 1% Triton X-100, 0.1% β-mercaptoethanol, 0.1 mmol/L phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, and 5 μg/mL aprotinin, pH 7.4. Homogenates were centrifuged at 4°C for 15 min at 14,000 g. The supernatants were then collected, and protein concentrations were determined via a protein assay kit (Bio-Rad, Hercules, CA). Samples with equal amounts of protein (30 μg) were loaded onto 12% polyacrylamide gels with 0.1% sodium dodecyl sulfate in the running buffer and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were then transferred onto nitrocellulose membranes. Nonspecific binding sites were blocked for 3 to 4 h at room temperature in a Tris-buffered saline solution containing 5% non-fat dry milk. The membranes were probed with primary antibodies against IBA1 (1:1000, Cell Signaling Technology, Danvers, MA). After washing, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase. Proteins were visualized with enhanced chemiluminescence reagents and blots were exposed to Hyperfilm. The results were analyzed with Kodak ID image analysis software. Band intensities were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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