MCL was isolated from Michelia compressa (Magnoliaceae), and HPLC purity was ≥99%. Its molecular weight is 248.32 KDa, and the molecular formula is C15H20O4 (Fig. S1). Primary Iba1 antibody was ordered from Wako (Japan). Anti-GFAP antibody was ordered from Abcam (Japan). Biotin-rabbit anti-mouse IgG and SYBR master mix were ordered from Life Technologies (USA). The second antibody and TRIzol were ordered from Invitrogen (USA). 3,3′-diaminobenzidine (DAB), thioflavin-s, LPS and BCA protein assay reagents were ordered from Sigma (Germany). Genomic DNA isolation solutions were ordered from Promega (USA). Agarose was ordered from Biowest (France). Bio-Plex Pro mouse chemokine panel 23-plex was ordered from Bio-Rad (USA). Reverse transcriptase was ordered from Takara (Japan). RIPA was ordered from Beyotime (China). A phosphatase inhibitor was ordered from Roche (Switzerland). AF488-labeled goat anti-rabbit antibody was ordered from Molecular Probes (USA).
Iba1 primary antibody
Manufactured by Fujifilm
Sourced in Japan
The Iba1 primary antibody is a laboratory reagent used for the detection and localization of the Iba1 protein, which is a marker for microglial cells in the central nervous system. The Iba1 antibody is commonly used in immunohistochemistry and immunofluorescence applications to study the distribution and activation state of microglial cells in various biological samples.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Vectastain abc hrp kit, by Vector Laboratories (2 mentions)
Sybr master mix, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Agarose, by Biowest (1 mentions)
Anti gfap antibody, by Abcam (1 mentions)
Bio plex pro mouse chemokine panel 23 plex, by Bio-Rad (1 mentions)
Biotin rabbit anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Axiovs 40 4, by Zeiss (1 mentions)
Cleaved caspase 3 primary antibody, by Cell Signaling Technology (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Triton x, by Merck Group (1 mentions)
Dimethylformamide, by Merck Group (1 mentions)
Piperidine, by Merck Group (1 mentions)
10 protocols using iba1 primary antibody
1
Isolation and Characterization of MCL from Michelia compressa
2
Quantifying Microglial Activation in Brain Tissue
3
Histological Analyses of Tumor Cell Apoptosis and Macrophages
4
PAMAM Dendrimer Bioconjugation and Cellular Imaging
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Hydroxyl‐terminated Generation 4 (G4) and Generation 6 (G6) poly(amidoamine) (PAMAM) dendrimers were purchased from Dendritech (Midland, MI). Cy3‐ and Cy5‐mono‐NHS esters were purchased from GE Healthcare (Chicago, IL). Benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N‐diisopropylethylamine (DIEA), dimethylformamide (DMF), Piperidine, dimethyl sulfoxide (DMSO), trimethylamine, 6‐Fmoc‐GABA‐OH, Triton X, and bovine serum albumin were purchased from Sigma‐Aldrich (St. Louis, MO). Fischer 344 rats were purchased from Harlan Bioproducts (Indianapolis, IN), and 9L gliosarcoma cells were purchased from the Brain Tumor Research Center of UC San Francisco (San Francisco, CA). C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and GL261 murine glioblastoma cells were purchased from the DTP/DCTD/NCI Tumor Repository (Bethesda, MD). RPMI, fetal bovine serum, penstrep antibiotic, l ‐glutamine, and normal goat serum (NGS) were purchased from ThermoFisher (Waltham, MA). Tris‐buffered saline (TBS) and phosphate buffered saline (PBS) were purchased from Corning (Corning, NY). Iba1 primary antibody was purchased from Wako Pure Chemical Corporation (Tokyo, Japan). Goat anti‐rabbit Alexafluor 488 secondary antibody was purchased from Invitrogen (Carlsbad, CA). NucBlue cell stain (DAPI) was purchased from Cell Signaling (Danvers, MA).
5
Immunohistochemical Analysis of Microglia in Mouse Brains
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Mice brains were removed and then soaked in 4% PFA at 4°C overnight, then dehydrated with a sucrose gradient of 20% and 30% respectively. Mice brains were cut into brain slices with a thickness of 20 µm with a cryostat (Leica, Germany). Brains and primary microglia cell were fixed with 4% PFA, washed with PBS, punched with 0.25% TritonX-100 and blocked with 4% BSA. Brains and primary microglia cell were stained by IBA1 primary antibody (Wako, Japan) and placed in a refrigerator at 4°C overnight. The brain slices were stained by fluorescence anti-rabbit/mouse secondary antibody (Invitrogen, United States), transferred to glass slides and mounted with DAPI-containing (Abcam, United Kingdom) mounting medium. Finally, brain slices were observed and photographed under an inverted fluorescence microscope (Olympus, Japan). Primary microglia cells were stained by DAPI (vector, United States) for nucleus. The state and purity of primary microglia were observed and photographed under an upright fluorescence microscope (Olympus, Japan).
6
Neuroinflammation in APP/APOE Mouse Model
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Human APOE3-KI and APOE4-KI mice were cross-bred with mice overexpressing mutant human APP (J20 line) to generate J20/APOE4-KI and J20/APOE3-KI mice, as we reported previously13 . All mouse lines were maintained on a C57Bl/6J background. Sex- and age-matched wildtype mice were used as controls. Brains were collected from female J20/APOE4-KI and J20/APOE3-KI mice (n=3 for each group) at 13 months of age. Brain sections were collected (30μm) from paraformaldehyde-fixed right hemibrains on a sliding microtome fitted with a freezing stage as described previously. Free-floating 30 μm sections were washed three times in PBS followed by blocking with 10% donkey serum in PBS for 1 h. Sections were incubated in PBS with 5% donkey serum and Iba1 primary antibody (1:500; Wako 019–19741) for 72 h at 4 °C. After primary antibody incubation, sections were washed once in PBS and incubated in the following secondary antibody and lipid droplet dye for 2 hr at RT: donkey anti-rabbit 555 (1:500; Invitrogen) and LipidSpot 488 (1:1000; Biotium). Sections were incubated in DAPI (1:2000; Thermo Fisher) for 10 min, then washed three times with PBS. Sections were mounted on microscope slides with ProLong Glass mounting media. Imaging was performed with a ZEISS LSM 900 confocal microscope ZEN 3.0 (Blue Edition) software.
7
Astrocyte and Microglia Activation in Brain
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To assess changes in the expression of reactive astrocytes and activated microglia, sections were incubated for 48 h with rabbit glial fibrillary acidic protein (GFAP) primary antibody (Dako; Z0334, 1:500 in incubation buffer) or with rabbit ionized calcium-binding adapter molecule 1 (Iba-1) primary antibody (Wako; 019-19741, 1:500 in incubation buffer), respectively. The sections then were washed and incubated with donkey anti-rabbit secondary antibody (Abcam; Alexa Fluor® 594 ab150064, 1:300 in incubation buffer). After rinsing with PBST, sections were mounted onto 2% gelatin-coated slides. The brain sections were observed using a Leica SP5 confocal microscope (Leica, Germany), using a ×40 magnification. For each brain, three to four sections were used to capture six to eight images from the hippocampus (CA3 and dentate gyrus) and cortex. Images were taken from both sides of the brain, and subsequently, were quantitively analyzed using the Imaris software (Bitplane AG, Zurich, Switzerland) to provide a mean value for each brain region.
8
Immunohistochemical and Immunofluorescence Analysis of Microglia
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All procedures were performed as previously described [43 (link), 44 ]. Briefly, mice were anesthetized by pentobarbital sodium and perfused with saline. Brains were fixed with 4% paraformaldehyde (w/v) for 7 days. Fixed brains were transferred to 30% sucrose solution for 3 days. Coronal sections were cut throughout the whole brain. In immunohistochemistry (IHC), brain slices were blocked with 10% goat serum (Abcom, ab7481) in PBS containing 0.2% Triton X-100 (Sigma, V900502), and incubated with Iba1 primary antibody (WAKO, 019-19741, 1:400), biotinylated goat anti-rabbit IgG, and streptavidin-conjugated horseradish peroxidase using VECTASTAIN® ABC-HRP kit (Vector Laboratories, PK-4000). Iba1 stains were visualized with 3,3′-diaminobenzidine (Sigma-Aldrich), scanned and analyzed by stereo investigator (MicroBrightfield). In immunofluorescence, brain slices were blocked, and incubated with primary and secondary antibodies as follow: Iba1 (Novus Biologicals, NB100-1028, 1:400), ASC (Cell Signaling Technology, D2W8U, 1:500), Alexa Fluor 546-conjugated (Invitrogen, A-11056, 1:400), and FITC-conjugated (Abcam, ab6798, 1:400). Hoechst 33258 (Sigma) were incubated for the visualization of nuclear morphology.
9
Immunohistochemical Detection of Iba1 in Liver
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Liver sections were de‐paraffinized with xylene and rehydrated by successive incubations in ethanol 100, 95, and 70% and water. Thereafter, slides were incubated in 10 mM sodium citrate buffer (pH 6) at sub‐boiling temperature for 10 min and cooled at room temperature for 30 min. Before staining, slides were washed three times with water and incubated in 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity and washed twice in water. Thereafter, slides were blocked in 5% goat serum in TBST. Iba1 primary antibody (Wako) was used to detect Iba1 expression. Later, we used the VectaStain ABC HRP Kit (Vector Labs) and diaminobenzidine to detect immunoreactivity. Finally, slides were counter‐stained with hematoxylin, dehydrated, and mounted for bright‐field microscopy.
10
Iba1 Immunohistochemistry of Microglia
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