The cerebella were dissected from mice and lysed in RIPA lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 0.2% SDS, phosphatase (Sigma) and protease inhibitors cocktail (Roche)). After three cycles of freeze and thaw, proteins were separated on a 12% or 15% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The following primary antibodies were used: anti-ATXN1 (rabbit 11NQ, Orr lab), anti-Iba1 (DAKO), anti-synapsin (Synaptic systems), anti-PSD95 (Biolegend), anti-TNF alpha (Abcam ab34674), and alpha-tubulin (mouse, Sigma). Signals from secondary antibodies linked to horseradish peroxidase (HRP) (GE Healthcare) were detected using Amersham ECL Western Blotting Detection Reagent (GE Healthcare) and ImageQuant LAS 4000 imager (GE Healthcare); protein levels were quantified using ImageQuant (GE healthcare) and ImageJ software. For tumor necrosis factor alpha (TNFα), we quantified the 20 kD band, and for synapsin, we quantified synapsin 2a band. Data was analyzed with one-way ANOVA followed by Bonferroni post hoc test and Student’s t test with Welch’s correction.
Anti iba1
Manufactured by Agilent Technologies
Sourced in Japan
Anti-Iba1 is a lab equipment product from Agilent Technologies. It is an antibody used for the detection of Iba1, a protein involved in the activation and regulation of macrophages and microglia. The core function of Anti-Iba1 is to facilitate the identification and analysis of Iba1-expressing cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
Anti synapsin, by Synaptic Systems (1 mentions)
Anti psd95, by BioLegend (1 mentions)
Anti tnf alpha, by Abcam (1 mentions)
Alpha tubulin mouse, by Merck Group (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Imagequant, by GE Healthcare (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
Anti cd31, by R&D Systems (1 mentions)
Anti collagen 4, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Anti gfap, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Axiovert 200m fluorescent microscope, by Zeiss (1 mentions)
Anti ikkβ, by Merck Group (1 mentions)
Anti neun, by Santa Cruz Biotechnology (1 mentions)
Anti gfp, by Santa Cruz Biotechnology (1 mentions)
Anti gfap, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti iba1
1
Western Blot Analysis of Cerebellar Proteins
2
Simultaneous Visualization of Microglia and Vasculature
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Brain hemispheres were removed from PBS-perfused mice, fixed in 4% paraformaldehyde (PFA) for 3 to 16 h, and cryopreserved in 30% sucrose until the tissue sank to the bottom of the tube. Brains were embedded in OCT freezing medium and sectioned into 25 μm-thick sagittal tissue sections for staining. Brain sections were stained with anti-IBA-1 (Dako, Japan) and either anti-CD31 (R&D Systems, Minneapolis, MN) or anti-collagen IV (EMD-Millipore, Germany). Sections were permeabilized for 1 h in permeabilization buffer (0.3% Triton X-100, 3% bovine serum albumin, 1× PBS) and incubated at room temperature for 16 to 48 h with primary antibodies diluted 1:500 in permeabilization buffer. Sections were washed with 1× PBS three times and incubated with secondary antibodies (Thermo Fisher) diluted in permeabilization buffer for 2 h to overnight. Sections were washed and mounted (with or without 4′,6-diamidino-2-phenylindole [DAPI]) for imaging on an SP8 confocal microscope (Leica).
3
Immunocytochemical Analysis of Neural Cells
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Immunocytochemical tests were carried out using an immunoassay kit (DAKO, EUA) according to the manufacturer’s protocol. For phenotypic characterization of primary cultures of neural cells, neurons were labeled with antibodies against the NeuN protein (anti-NeuN) (DAKO, EUA), astrocytes were labeled with antibodies against glial fibrillary acidic protein (anti-GFAP) (Thermo Scientific, EUA), and microglia were labeled with antibodies against adapter-ionized (AIP) calcium-binding molecule 1 (anti-IBA 1) (DAKO). The immunohistochemical pattern of cultures was obtained by calculating the percentage of labeled cells (NeuN, GFAP, and IBA 1) in relation to the total number of cells stained, as previously reported by Kádár et al. ( 6 (link) )The cells were analyzed using an inverted microscope. Control wells not treated with the primary antibodies were included in the test.
4
Immunohistochemical Profiling of Neural Markers
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Sections were penetrated with 0.1% Triton X-100 (2 × 10 min at 25°C); washed in PBS (3 × 5 min at 25°C); blocked with 10% goat or donkey serum (30 min at 25°C); treated with 1:250 anti-IKKβ (Millipore), 1:500 anti-NeuN (Santa Cruz Biotechnology), 1:300 anti-GFAP (Santa Cruz Biotechnology), 1:1000 anti-IBA1 (Dako), and 1:1000 anti-GFP (Santa Cruz Biotechnology) antibodies (4°C overnight); washed in PBS (3 × 10 min at 25°C); and then subjected to reaction with fluorescence-conjugated secondary antibodies of 1:1000 Alexa Fluor 488 and 1:1000 Alexa Fluor 568 (Invitrogen; 2 h at 25°C); and rinsed with PBS (3 × 10 min at 25°C). The sections were mounted on slides using sterile 0.2% gelatin and DAPI mounting media (Vector Laboratories) and coverslipped. Images were taken using either an Axiovert 200M Fluorescent Microscope (Zeiss) equipped with a 20× objective or an LSM 710 Confocal Microscope (Zeiss) equipped with a 63× objective. For the immunohistochemistry, the following two sets of control experiments were performed to test specificity: (1) replacement of the primary antibody with only the serum of the appropriate species; and (2) omission of secondary antibodies. No immunostaining was detected under either of these conditions.
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