All stainings were performed on 10 μm fresh-frozen sections. Oil red O (ORO) staining as well as hematoxylin and eosin (HE) staining of liver sections was performed to define liver histopathologies. Immunoflorescent (IF) stainings of mouse liver sections were performed using the following primary antibodies: anti-mouse/human ApoE (ab52607; Abcam); anti-mouse C1q (ab182451; Abcam); anti-mouse CLEC4F (ab2608299; Invitrogen); anti-mouse CD68 (FA11; Serotec); anti-mouse CD31 (ab553370; BD PharMingen); anti-human C3 (A213; ComplementTech); anti-mouse C4 (HM1046; Hycult Biotech); anti-mouse C5 (ab11898, Abcam). We had previously established the specificity of IF microscopy in mouse and human tissues including no primary antibody negative controls, isotype antibody controls, ApoE knockout mouse tissue (i.e., anti-ApoE antibodies) (1 (link)). IF stainings of human sections were performed using the following antibodies: anti-mouse/human ApoE (ab52607; Abcam); anti-human C1q (ab71089; Abcam); anti-human CD68 (EMB11; DAKO); anti-human C5 (A220; ComplementTech). Hepatitis sections as well as the appropriate control tissues were fixated with Delaunay solution prior to IF staining. Stained sections were analyzed using a Leica confocal microscope (SP8, Leica, Germany) using Leica Application Suite (Leica) and ImageJ software.
Ab52607
Manufactured by Abcam
Sourced in United States
Ab52607 is a solid-phase microplate-based enzyme-linked immunosorbent assay (ELISA) kit designed for the quantitative measurement of human interleukin-6 (IL-6) in cell culture supernatants, serum, and plasma samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab71089, by Abcam (2 mentions)
Leica confocal microscope, by Leica camera (2 mentions)
Ab209234, by Abcam (2 mentions)
Ab129728, by Abcam (1 mentions)
Ab55847, by Abcam (1 mentions)
Enhanced chemiluminescence chromogenic substrate, by GE Healthcare (1 mentions)
Pa5 42049, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab8245, by Abcam (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Ab182451, by Abcam (1 mentions)
Leica application suite, by Leica camera (1 mentions)
Ab183597, by Abcam (1 mentions)
Duolink pla kit, by Merck Group (1 mentions)
Ab292, by Abcam (1 mentions)
Ab1906, by Abcam (1 mentions)
Ab23792, by Abcam (1 mentions)
Ab214421, by Abcam (1 mentions)
Mouse α gfp, by Takara Bio (1 mentions)
Goat α apoe, by Merck Group (1 mentions)
8 protocols using ab52607
1
Comprehensive Liver Histopathology and Immunofluorescence Analysis
2
Protein Expression Analysis by Western Blot
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According to the results of proteome sequencing, WB was performed on the selected eight protein targets. Tissues were collected and homogenized using RIPA buffer (Beyotime Institute of Biotechnology). Equal amounts of total protein (30 µg/lane) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, USA). The primary antibodies were anti-IGFBP2 (ab227881, 1:1,000, Abcam, USA), anti-TPM3 (ab190993, 1:1,000, Abcam, USA), anti-ALDOB (ab129728, 1:1,000, Abcam, USA), anti-FAH (PA5-42049, 1:1,000, Invitrogen, USA), anti-SOD3 (14316-1-AP, 1:1,000, Proteintech, China), anti-SPARC (ab55847, 1:1,000, Abcam, USA), anti-APOE (ab52607, 1:1,000, Abcam, USA), anti-RBP4 (ab154914, 1:1,000, Abcam, USA), and anti-GAPDH (ab8245, 1:5,000, Abcam, USA). And the secondary antibody goat was anti-mouse IgG (PAB31797; Abnova, China). The bands were then developed using enhanced chemiluminescence chromogenic substrate (GE Healthcare, UK) and analyzed by the Image J software. GAPDH was used as control in this study.
3
In situ PLA for Protein-Protein Interactions
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Protein-protein interaction ex vivo in mouse and human tissues in situ were performed using the Duolink® PLA kit (DUO92101 SIGMA) as previously described in (1 (link)): briefly, sections were stained with rabbit anti mouse ApoE (ab183597, Abcam) and mouse anti-C1q (HM1096BT, Hycult) for mouse liver tissues and with rabbit anti-human ApoE (ab52607, Abcam) and mouse anti-human C1q (ab71089, Abcam) for human livers. No or only one primary antibody were used as controls. PLA signals were detected according to the manufacturer’s protocol. A Leica confocal microscope (SP8, Leica, Germany) equipped with a 96x or 100x oil objective (NA 1.4) was used for imaging.
4
Antibody Identification and Characterization Protocol
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Antibodies and their sources were as follows: anti-CD63 (ab23792, Abcam, Paris, France) (1:200 dilution for WB and EM), anti-CD9 was a kind gift of Eric Rubinstein (Inserm, U935, Villejuif, France) (dilution 1:500 for WB and 1:200 for EM), anti-ApoE (ab52607 or ab1906, Abcam, Paris, France) (dilution 1:500 for WB), anti-fibronectin (F3648, Sigma-Alrich, Merck, Saint Quentin Fallavier, France) (dilution 1:1000 for WB), anti-collagen (ab292, Abcam, Paris, France) (dilution 1:1000 for WB), horseradish peroxidase (HRP)-conjugated goat polyclonal antibodies to rabbit IgG and to mouse IgG (Abcam, Paris, France) (dilution 1:10000 for WB), protein A conjugated to 10 nm gold particles (Cell Microscopy Center, Utrecht University Hospital, Utrecht, The Netherlands) (PAG; dilution 1:50 for EM).
Rat tail collagen I (Catalog Number 354236) was from Corning, Sigma-Aldrich, Merck, Saint Quentin Fallavier, France.
Rat tail collagen I (Catalog Number 354236) was from Corning, Sigma-Aldrich, Merck, Saint Quentin Fallavier, France.
5
Immunofluorescence Assay for Protein Expression
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mIF assay was implemented according to the methods described previously (55 (link)). The primary antibodies included anti-APOE (1:50, ab52607, Abcam), anti-BGN (1:50, ab209234, Abcam), anti-CTLA4 (1:200, bs-1179R, Bioss, Woburn, MA, USA), and anti-PDCD1 (1:100, ab214421, Abcam). Using ImageJ software, we evaluated the expression of protein levels in the tissue sections on the basis of the corrected total cell fluorescence (CTCF).
6
Antibody Characterization for Immunodetection
7
Immunohistochemistry Assay Protocol for Protein Expression Analysis
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IHC assay was carried out according to the methods described previously (20 (link)). To ensure the consistency of the analysis, all IHC assays were performed using the same type of tissue chips. The primary antibodies included anti-APOE (1:800, ab52607, Abcam, Cambridge, UK), anti-AHSG (1:1,000, ab187051, Abcam), anti-BGN (1:5,000, ab209234, Abcam), anti-SLC2A4 (1:500, 66846-1-Ig, ProteinTech, Chicago, IL, USA), and anti-CYP2D6 (1:500, ab185625, Abcam). We evaluated the expression of protein levels in the tissue sections based on the IRS: 0–1 indicating negative staining, 2–3 indicating mild staining, 4–8 indicating moderate staining, and 9–12 indicating strong positive staining.
8
Quantifying ApoE Levels in Cell Media
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Semiquantitative Western blot analysis was performed on cell lysates from HepG2 cells using antibodies specific to either the N-terminus (ab51015, Abcam) or the C-terminus (ab52607, Abcam) of ApoE. β Actin (ACTB) antibody was used as a loading control. Relative quantification of the HepG2 cell lysate was measured by densitometry using a standard curve method. We quantified ApoE levels in culture medium using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Mabtech, #3712-1HP-2). Media was collected from three independent 5-aza-Dc treated and three untreated HepG2, LN229, and SHSY5Y cell cultures (18 total). Culture medium was refreshed at 48 hrs and collected at 72 hrs. Media was cleared by centrifuging at 1500 rpm for 10 min at 4°C, supernatant was collected and frozen in two aliquots. Due to the high level of ApoE expression in HepG2 cells, media was diluted 1:200 in Apo ELISA buffer prior to assaying. Media from LN229 and SHSY5Y cultures was not diluted. All samples were run in duplicate and concentrations were averaged to account for technical variation. ApoE levels in the media were normalized by total cell number to account for cell death caused by 5-aza-dC treatment.
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