The Western blot was performed as previously described [25 (link)]. Liver protein extracts were obtained by lyses homogenized tissue in Ripa buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with protease inhibitors (Roche Diagnostics, Mannheim, Germany). Protein samples (40 μg) were resolved on 10% or 12% Tris-HCl polyacrylamide gels and subsequently transferred to a nitrocellulose membrane. Membranes were probed with commercially available rabbit polyclonal anti-ApoE (1:500 dilution), anti-GRP-78 (1:500) (Abcam, Cambridge, MA, USA), anti-β-tubulin (1:200) (Cell Signaling, Danvers, MA, USA), anti- ERP29 and anti-α-tubulin (1:2000), anti-SREBP -1c and anti-SOD2 (1:500) antibodies (Abcam, Cambridge, MA, USA), followed by HRP-conjugated anti-rabbit antibody (1:10000) and ECL Plus detection reagents (GE Biosciences, Piscataway, NJ, USA). Relative ApoE, GRP-78, β-tubulin, α-tubulin, ERP29, SREBP and SOD2 band densities were determined by densitometrical analysis using the Image Studio Lite software from LI-COR Corporate Offices-US (Lincoln, Nebraska USA). In all instances, density values of bands were corrected by subtraction of the background values. The results were expressed as the ratio of ApoE, GRP-78, ERP29 and SOD2 to that of β-tubulin and SREBP to that of α-tubulin our β-tubulin.
Anti apoe
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-ApoE is a laboratory reagent used for the detection and quantification of apolipoprotein E (ApoE) in biological samples. ApoE is a protein involved in the metabolism and transport of cholesterol and triglycerides. This product can be used in various research applications that require the analysis of ApoE levels.
Automatically generated - may contain errors
Lab products found in correlation
Anti iba1, by Fujifilm (3 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Ecl plus detection reagent, by GE Healthcare (1 mentions)
Anti srebp 1c, by Abcam (1 mentions)
Anti sod2, by Abcam (1 mentions)
Anti grp78, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Anti nrf2, by Abcam (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Goat serum, by Boster Bio (1 mentions)
Anti apoa 1, by Santa Cruz Biotechnology (1 mentions)
Protein g sepharose 4 fast flow beads, by GE Healthcare (1 mentions)
Anti synapsin 1, by Abcam (1 mentions)
Anti syntaxin 1b, by Synaptic Systems (1 mentions)
Anti synaptophysin, by Santa Cruz Biotechnology (1 mentions)
Anti human cd68, by Agilent Technologies (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemiluminescence imaging system, by Bio-Rad (1 mentions)
Phosphatase inhibitor, by Boster Bio (1 mentions)
10 protocols using anti apoe
1
Western Blot Analysis of Liver Proteins
2
Immunofluorescence Staining of ApoE and Nrf2
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For immunofluorescence staining, tissue sections were deparaffinized by xylene, rinsed, and blocked with goat serum (Boster Biological Technology, Pleasanton, California, USA) at 37°C in a humidified atmosphere for 1 h. Then sections were incubated overnight at 4°C with rabbit monoclonal anti‐ApoE (1:400; Abcam, Cambridge, UK) or rabbit polyclonal anti‐Nrf2 (1:400; Abcam). The sections were rinsed with 0.01 M PBS and incubated with goat IgG conjugated to Alexa 492 (1:200; Earthox, Shanghai, China) for 1 h at 37°C in a dark humidified chamber. Finally, nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (Beyotime Bio, Shanghai, China) for 5 min. After a final rinse, coverslips were mounted with an antifade mounting medium (Beyotime Bio), and sections were observed under a fluorescence microscope.
3
Reverse Co-Immunoprecipitation of Synaptic Proteins
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For reverse co-IPs, protein G-Sepharose 4 Fast Flow beads (GE Healthcare) were pre-bound with 4 μg of the following antibodies, respectively: anti-mitochondrial creatine kinase U-type (MtCK, Abcam, San Francisco, catalog no. ab76506); anti-β-synuclein (Abcam, catalog no. ab76111); anti-apoE (Abcam, catalog no. ab1906); anti-apoAI (Santa Cruz Biotechnology, Dallas, TX, catalog no. sc-30089); anti-syntaxin 1B (Synaptic Systems, Göttingen, Germany, catalog no. 110402); anti-synapsin 1 (Abcam, catalog no. ab8); anti-synaptogyrin-3 (Santa Cruz Biotechnology, catalog no. sc-68936); anti-synaptophysin (Santa Cruz Biotechnology, catalog no. sc-9116); and anti-synaptotagmin (Millipore, catalog no. MAB5200). These samples were then incubated with 1 mg of lysate at 4 °C overnight. To verify the binding preference of Tau isoforms, dephosphorylated lysates were used adapting a dephosphorylation protocol obtained from New England Biolabs. Beads were washed extensively with IP washing buffer and eluted in 2× SDS-PAGE sample buffer for subsequent Western blot analysis. Blots were probed with the following antibodies: anti-creatine kinase U-type; mitochondrial (MtCK); anti-β-synuclein; anti-apoE; anti-apoAI; anti-syntaxin 1B; anti-synapsin 1; anti-synaptogyrin-3; anti-synaptophysin; anti-synaptotagmin, and anti-Tau. For quantification, ImageJ was used.
4
Immunofluorescence Imaging of CD68 and ApoE
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Immunofluorescence was performed on 2-μm-thick formalin-fixed paraffin-embedded CLM sections using the Opal Kit (Perkin Elmer) following the manufacturer’s recommendations. The following primary antibodies were used: anti-human CD68 (Dako; clone KP.1, diluted 1:1,000) and anti-ApoE (Abcam; clone EPR19392, diluted 1:1,500). Representative images were acquired using an SP8II confocal microscope (Leica).
5
Western Blot Analysis of Brain Protein Expression
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Brain hemispheres of mice were homogenized with RIPA (Beyotime, Haimen, Jiangsu, China) plus protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitors (Boster, Wuhan, Hubei, China). Prepared protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were probed overnight at 4°C with the following primary antibodies: anti-ApoE (1 : 500, Abcam, Cambridge, USA), anti-Iba-1 (1 : 500, Wako, Osaka, Japan), anti-P-JNK (1 : 1000, CST, Danvers, MA, USA), anti-JNK (1 : 500, Santa Cruz, Dallas, TX, USA), anti-P-c-Jun (1 : 1000, CST, Danvers, MA, USA), and anti-c-Jun (1 : 1000, CST, Danvers, MA, USA) followed by incubation with secondary antibodies conjugated with horseradish peroxidase. The bands were revealed using an ECL western blotting kit (Thermo Scientific, Pittsburgh, PA, USA) and photographed with a chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). The amount of protein in each band was quantified using Image Lab Software (Bio-Rad, Hercules, CA, USA).
6
Western Blot Analysis of ApoE and ApoC1
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A total of 10 uL of each fraction was analyzed by Western blot using anti-apoE (52607-1:5000; Abcam, Cambridge, UK) and anti-apoC1 (198288-1:1000; Abcam). Proteins were separated on 4%–12% Bis-Tris NuPAGE polyacrylamide gels (Thermo Scientific) and transferred to 0.2-μm nitrocellulose membranes. Membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween 100 and probed with specific primary antibodies. After secondary antibody staining with peroxidase column purified donkey anti-rat IgG (H + L; 1:10,000), blots were visualized using SuperSignal West Dura reagent (ThermoFisher Scientific).
7
Immunostaining of Brain Slices
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Immunostaining for brain slices was performed as described previously [7 (link)], in which the primary antibodies were also listed. New antibodies used in this study include: thioflavin S (MCE, HY-D0972, Monmouth Junction, NJ), anti-C12orf34 (aggregatin) (1:100, Abcam, ab122626, Boston, MA), anti-Aβ (1:100, Proteintech, 60342-1-Ig), anti-synaptophysin (1:100, Proteintech, 17785-1-AP), anti-AT8 (1:100, Invitrogen, MN1020, Carlsbad, CA), anti-ApoE (1:100, Abcam, ab20874), anti-pTau-S396 (1:100, Thermo Fisher Scientific, 44-752G), and anti-Secretogranin III (1:100, Proteintech, 10954-1-AP).
8
Quantitative Western Blot Analysis of Key Targets
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Western blot analysis was performed as previously described [22] (link). After sample preparation, equal amounts of a sample protein (50 μg) were loaded onto an SDS-PAGE gel. After electrophoresis, the samples were transferred onto a nitrocellulose membrane. Then, the membrane was blocked for 2 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-apoE (1:500, Abcam, Cambridge, MA, USA), anti-Iba-1 (1:1000, Wako, USA), anti-APP (1:1500, Abcam, Cambridge, MA, USA), anti-myelin basic protein (MBP, 1:1000, Abcam, Cambridge, MA, USA), anti-LRP1 (1:1000, Abcam, Cambridge, MA, USA), anti-Shc1 (1:2000, Abcam, Cambridge, MA, USA), anti-PI3K (1:1000, Cell signaling, USA), anti-Akt (1:1000, Cell signaling, Danvers, MA, USA), anti-phospho-Akt (p-Akt, 1:1000, Cell signaling, Danvers, MA, USA), anti-CD16 (1:1500, Santa Cruz, Dallas, TX, USA), anti-iNOS (1:500, Abcam, Cambridge, MA, USA), anti-CD206 (1:1500, Santa Cruz, Dallas, TX, USA), and anti-β-actin (1:5000, Santa Cruz, Dallas, TX, USA). Appropriate secondary antibodies (1:5000, Santa Cruz, Dallas, TX, USA) were selected to incubate with the membrane for 2 h at room temperature. Then, blot bands were visualized with an ECL reagent (Amersham Biosciences UK Ltd., PA, USA). Non-saturated bands were selected to perform densitometry quantification using Image J software (Image J 1.51, NIH, USA).
9
Immunohistochemical Analysis of Brain Samples
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Brain samples were fixed with 4% paraformaldehyde for 4 h, followed by overnight immersion in phosphate buffer containing 30% sucrose. The brain samples then were embedded in OCT solution, and coronal frozen sections (10 μm) were prepared. The sections were incubated with primary antibodies at 4°C overnight, including anti-β-APP (1 : 100, Abcam, Cambridge, USA), anti-ApoE (1 : 100, Abcam, Cambridge, USA), anti-NeuN (Abcam, Cambridge, USA), anti-Iba-1 (1 : 200, Wako, Osaka, Japan), and anti-P-JNK (Abcam, Cambridge, USA). Sections were then incubated with DyLight 488-conjugated goat anti-rabbit and DyLight 549-conjugated goat anti-mouse secondary antibodies (Abbkine, Redlands, USA). DAPI was used for nuclear staining. The number of positive cells was counted with Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, USA).
10
Protein expression analysis of mouse cortex
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