Brain sections (40 µm) were blocked with TBS-TS for 30 min at room temperature and incubated with the primary antibodies, i.e., anti-GFAP (mouse polyclonal; 1:500, Cell Signaling, MA) and anti-ionized calcium-binding protein (Iba-1) antibody (rabbit polyclonal; 1:500, Wako, Tokyo, Japan) in TBS-TS at 4 °C overnight. The sections were then washed with TBS, incubated with anti-mouse IgG labeled with Alexa Fluor 488 (3 μL/mL) and anti-rabbit IgG labeled with Alexa Flour 568 (3 μL/mL) for 3 h at room temperature. Images were obtained using a ZEISS LSM800 confocal microscope (Oberkochen, Germany). Two or three sections per mouse containing SN or STR were taken, and the fluorescence intensity over the full range of images in both the left and right sides was measured by Image J software, averaged, and considered as n = 1.
Iba1 antibody
Manufactured by Fujifilm
Sourced in Japan, United States
The Iba1 antibody is a research-use laboratory instrument produced by Fujifilm. It is a protein marker that is commonly used in immunohistochemistry and Western blotting applications to detect macrophages and microglia in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm800 confocal microscope, by Zeiss (4 mentions)
Gfap antibody, by Merck Group (4 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Confocal microscope, by Leica (3 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (2 mentions)
Abc reagent, by Vector Laboratories (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Lsm 5 pascal, by Zeiss (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Abc kit, by Vector Laboratories (2 mentions)
Fluorescence microscope, by Nikon (2 mentions)
Micro slides superfrost plus glass slides, by Electron Microscopy Sciences (2 mentions)
Alexa 488 anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Cy3 anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Fluoromount g, by Electron Microscopy Sciences (2 mentions)
Monoclonal anti aβ 6e10, by Fortrea (2 mentions)
Vibratome, by Leica (2 mentions)
Cryostat, by Leica (2 mentions)
Lsm 510, by Zeiss (2 mentions)
80 protocols using iba1 antibody
1
Immunostaining of Brain Sections
2
Immunofluorescent Analysis of Retinal Microglia
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The mice were anesthetized with the IP injection of 1.5% sodium pentobarbital. Eyes were dissected out and post-fixed in 4% Paraformaldehyde solution (PFA) at 4°C for 1 h. For frozen sections, tissues were immersed in 10% and 20% sucrose for 30 min and in 30% sucrose overnight before sectioning in a cryostat with section thickness of 10 μm for retina. Before staining, retinal sections were incubated with 5% BSA and 1% Triton X-100 in Phosphate-buffered saline (PBS) for 1 h at room temperature, rinsed with the blocking solution, put into the blocking solution containing the primary Ionized calcium-binding adapter molecule 1 (IBA-1) antibody (1:500; Wako, cat. no. 019-19741, Japan), and incubated in a humidified box at 4°C for 12 to 16 h. After several washes, sections were incubated with the Alexa Fluor 488 secondary antibody (1:500, Thermo Fisher Scientific, USA, cat. no. A-11034). In addition, nuclei were stained with Hoechst 33342 (Sangon Biotech, USA, cat. no. E607302). The immunofluorescent images were obtained using a confocal microscope (Leica, Germany).
3
Immunofluorescent Imaging of Microglial Cells
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Mice were deeply anesthetized with pentobarbital and transcardially perfused with PBS and then 3.2% paraformaldehyde. Brains were postfixed in 3.2% paraformaldehyde for 48 h. Fixed brains were sectioned (40 μm) using a vibratome (Leica). Sections were blocked with 3% goat serum. Sections were washed and incubated with a rabbit anti-mouse ionized calcium binding adaptor molecule 1 (Iba-1) antibody (Wako Chemicals, USA), overnight at 4°C, followed by incubation with the appropriate fluorescent-conjugated secondary antibody (Invitrogen). Slices were then mounted on glass slices with Fluoroprep (Dako) containing 1 mg/ml Hoescht, a fluorescent specific DNA dye (Invitrogen). Each fluorochrome was independently captured with an FV10i scanning confocal microscope (Olympus, France). Quantification of Iba-1 staining was performed using ImageJ software (NIH). The software generated fluorescence intensity values by tracing the region-of-interest (ROI). Arbitrary units were defined in terms of strength of fluorescent signal. The final intensity values were calculated by subtracting the area of the selected region multiplied by the background fluorescence from the fluorescence intensity of the region-of-interest (ROI): fluorescence intensity (arbitrary units) = fluorescence intensity of ROI − (area of selected region × mean fluorescence of background).
4
Neuropathology Analysis in SOD1 Mice
5
Murine Neuroinflammation Immunostaining Protocol
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We obtained murine recombinant IL-33 (580506), AF647-conjugated Aβ (clone: 6E10) antibody (803021), APC-conjugated MHC-II (clone: M5/114.15.2) antibody (107614), FITC-conjugated VCAM1 (clone: MVCAM.A) antibody (105706) and MHC-II (I-A/I-E) (clone: M5/114.15.2) antibody (107601) from BioLegend. We obtained ICAM1-neutralizing (clone: YN1/1.7.4) antibody (BE0020)75 (link),76 (link) and VCAM1-neutralizing (clone: M/K-2.7) antibody (BE0027)69 (link),77 (link) from Bio X Cell. We obtained AF488-conjugated CD11b (clone: M1/70) antibody (53-0112-82), APC-conjugated CD11b (clone: M1/70) antibody (17-0112-83) and biotinylated CD11b (clone: M1/70) antibody (13-0112-82) from eBioscience. ApoE-neutralizing (clone: HJ6.3) antibody was a gift from D. Holtzman36 (link). We obtained DAPI (D3571) from Life Technologies, and mouse ITGB2 recombinant protein (LS-G14036 -10) was from LSBio. We obtained mouse ApoE recombinant protein (MBS955382) from MyBioSource as well as CCR7 neutralizing (clone: 4B12) antibody (MAB3477)78 (link) and VCAM1 antibody (BBA5) from R&D Systems. We obtained recombinant mouse CD44 protein (53953-M08H) from Sino Biological, MeX04 (4920) from Tocris Bioscience and Iba1 antibody (019-19741) from Wako.
6
Immunofluorescence Analysis of Microglia and Astrocytes
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Paraffin-embedded sections were deparaffinised according to standard procedures. The sections were then incubated in citrate buffer (pH 6.0) in a microwave oven for antigen retrieval. After blocking with 3% normal goat serum, sections were incubated in a solution of Iba-1 antibody (1/1000, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and GFAP antibody (1/50, Cell Signaling Technology, Tokyo, Japan) or in a control solution of normal rabbit IgG and normal mouse IgG overnight at 4 °C. After washing, sections were incubated with Alexa594-conjugated anti-rabbit IgG at room temperature for 30 min. After washing, the sections were covered with mounting media, including 4′,6-diamidino-2-phenylindole (DAPI; VECTASHIELD, Funakoshi, Tokyo). Iba-1 immunoreactivity data were obtained using a macro on an ImageJ.
7
Microglial Morphology Analysis in CNS
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Following sacrifice, brain tissue was dissected and fixed in 10% formalin overnight before paraffin embedding. Immunohistochemical staining was completed at the UMMS Morphology Core using anti-ionized calcium-binding adapter molecule (IBA1) antibody (Wako; 1:1000) and subsequently labeled with streptavidin-biotin immunoenzymatic antigen for detection with 3,3′-diaminobenzidine (DAB) (UltraVision Mouse Tissue Detection System Anti-Mouse HRP/DAB; Lab Vision). Images were acquired from the described CNS areas by light microscopy (cortex; CA1, CA3, and DG of the hippocampus) at × 40 magnification for process length and cell body size measurements of microglia using ImageJ. Cell process length for each microglial cell was measured by tracing all extensions off of the soma to their distal termination using ImageJ’s freehand measuring tool. For each microglia, the length of all processes was summed to obtain the total cell process length. The soma area was measured by tracing the perimeter of the cell body and measuring the contained area using ImageJ’s freehand tracer and the area measurement function. Microglia were analyzed from five to nine images taken randomly from each CNS region from each mouse. The investigator was blinded to the sample groups during staining, image acquisition, and ImageJ analysis. IBA1 positivity was measured using the Color Deconvolution plug-in in ImageJ.
8
Immunohistochemical Analysis of Brain Tissue
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Immunohistochemistry on paraffin-embedded sections was conducted, as described previously (Sahab-Negah et al. 2020 ). Briefly, brain tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The thickness of the tissues was cut into 5-μm sections. After deparaffinization, brain sections were boiled in 10 mM citric acid buffer (pH = 6) for 10 min. Sections were incubated with normal goat serum (Sigma, Germany) containing 0.3% Triton-X-100 in PBS. Sections were then incubated with primary antibodies, including mouse anti-glial fibrillary acidic protein (GFAP), a marker for reactive astrocytes (1:500; Abcam, UK); rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) antibody (1:1000; Wako) to encounter microglia at 4 °C overnight. Horseradish-peroxidase (HRP)–conjugated anti-mouse antibody (1:100; Abcam) and anti-rabbit antibody (1:500, Abcam) was added to sections at room temperature for 1 h. For negative controls, primary antibodies were deleted. The sections were visualized by 3,3′-diaminobenzidine and analyzed with a bright field microscope.
9
Immunofluorescence Imaging of Mouse Glioma
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The immunofluorescence assay was performed as previously described.62 (link) Briefly, the frozen slice of mouse glioma tissues were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 20 min, sealed with 10% BSA (BSA) in room temperature for 30 min, and incubated with IBA1 antibody (1:500; Wako, Japan; cat. 019–19,741) in a wet box overnight at 4°C in turn. After that, samples were washed three times with PBST and incubated with R-phycoerythrin-conjugated secondary antibody (1:200; 111-116-144; Jackson Laboratory) for 1 h at 37°C. Finally, these samples were stained with DAPI for 5 min at room temperature, and images were acquired using a fluorescent-microscope imaging system.
10
Iba-1 Antibody: Selective Microglial Marker
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