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 tnf alpha
Manufactured by Abcam
Anti-TNF alpha is a lab equipment product that neutralizes the biological activity of the cytokine tumor necrosis factor alpha (TNF-alpha). It functions by binding to and inhibiting the action of TNF-alpha, a key mediator of inflammation.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
Anti iba1, by Agilent Technologies (1 mentions)
Anti synapsin, by Synaptic Systems (1 mentions)
Anti psd95, by BioLegend (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)
Appropriate secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Imagej 4, by Media Cybernetics (1 mentions)
Ecl plus chemiluminescence kit, by Cytiva (1 mentions)
Anti nrf2, by Abcam (1 mentions)
Anti chemerin, by Abcam (1 mentions)
Anti ampkα, by Cell Signaling Technology (1 mentions)
Anti ccrl2, by Abcam (1 mentions)
Anti il1 beta, by Abcam (1 mentions)
Anti phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
Anti il 6, by Abcam (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
4 protocols using anti tnf alpha
1
Western Blot Analysis of Cerebellar Proteins
2
Comprehensive Protein Analysis via Western Blot
3
Fluorescent IHC for DH TNF-α in DG
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Experiment 1 used fluorescent immunohistochemistry (IHC) to examine alterations in DH TNF-α in the DG. The IHC protocol used here has been described previously [19 (link), 21 (link)]. Briefly, tissue sections were washed three times for 10 min in 0.1M phosphate buffer (PB, pH = 7.4), followed by a 1-h incubation in 5% normal goat serum (NGS) and 0.5% TritonX100 in 0.1M PB at room temperature. Tissue was incubated in primary antibody:rabbit anti-TNF alpha (1:1000, Abcam, Cambridge, MA, Cat# ab66579), 5% NGS, and 0.5% TritonX100 in 0.1MPB overnight at 4°C, washed three times for 10 min in 0.1M PB, and incubated in secondary antibody:goat anti-rabbit Alexa Fluor-488 (1:1000, ThermoFisher Scientific, Waltham, MA, Cat #A11008), 5% NGS, and 0.5% TritonX100 in 0.1M PB for 1 h at room temperature. Primary antibodies were verified by no primary control stains. Sections were mounted onto SuperFrost Plus slides (Fisher Scientific, Pittsburgh, PA) using Vectashield with DAPI hardset mounting medium (Vector Laboratories, Burlingame, CA).
4
Inflammatory Biomarkers in Intracerebral Hemorrhage
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Animals were sacrificed to extract the brain at 5 days after ICH and whole brains were divided into ipsilateral (infarcted) and contralateral hemispheres, which were frozen in liquid nitrogen. Brain tissues from each group were homogenized in Neuronal Protein Extraction Reagent (Pierce), which included 1× protease and phosphatase inhibitor (Pierce). Protein concentrations were determined using BCA Protein Assay Kit (Pierce). The samples were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Amersham) that were blocked in 5% non-fat dried milk (NFDM) and incubated with anti-COX2 (1:1000; abcam), anti-TNF alpha (1:1000; abcam), anti-CD11b (1:500; BD Pharmingen), and anti-myeloperoxidase (1: 1000; abcam) and β-actin. Membranes were incubated for 1 hour with a secondary antibody and the intensity of all protein bands was quantified by densitometry using the Bio-image Processing System (GenDix). Densitometric values were normalized to those of beta-actin, used as an internal control.
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