Fresh mice skins were fixed with 4% formalin and embedded with paraffin. For histopathology analysis, tissue sections were stained with H&E. For immunohistochemistry staining, the slides were blocked following the manufacturer’s instructions (BIOTnA biotech) and incubated with anti-NS3 Ab (GTX124252, GeneTex, Inc, Irvine, CA) or anti-c1q Ab (ab 71089, Abcam, Cambridge, MA) at 4 °C overnight. After washing with PBS, the slides were incubated with HRP-labeled secondary antibody for 30 min at room temperature and then loaded onto 3,3′-diaminobenzidine (DAB) or HRP green mixed reagent for 1–5 min. Hematoxylin counterstain was applied for 2 min. The quantification of DAB staining was determined by ImageJ software.
Ab71089
Manufactured by Abcam
Sourced in United States
Ab71089 is a primary antibody that recognizes the H2A histone family. It is designed for use in various research applications, including immunohistochemistry and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Ab52607, by Abcam (2 mentions)
Leica confocal microscope, by Leica camera (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Gtx124252, by GeneTex (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)
Ab11877, by Abcam (1 mentions)
Ab37230, by Abcam (1 mentions)
Ab186834, by Abcam (1 mentions)
Ab23457, by Abcam (1 mentions)
Ab192577, by Abcam (1 mentions)
Ab118820, by Abcam (1 mentions)
Mayer s hematoxylin, by Solarbio (1 mentions)
Zli 9018, by ZSGB-BIO (1 mentions)
Ab7260, by Abcam (1 mentions)
Startingblock t20 tbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
9 protocols using ab71089
1
Histopathological and Immunohistochemical Analysis
2
Comprehensive Liver Histopathology and Immunofluorescence Analysis
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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.
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
Immunohistochemical Analysis of C1q in SCI
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At 72 hrs post-injury, the SCI site was extracted and then fixed in 4% paraformaldehyde for 24 hrs, embedded in paraffin and sliced into 5 µm thick sections. Slides were dewaxed, incubated overnight at 4°C with primary antibody C1q (ab71089, Abcam), then blocked with secondary antibody and visualized with DAB. Nuclei were counterstained with hematoxylin. Sections were observed using a histologic microscope (OLYMPUS-BX51) and counting was performed in a blinded manner.
5
Immunohistochemical Analysis of Complement System
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Four micrometers of formaldehyde‐fixed pleural tissue were dewaxed and rehydrated with an alcohol gradient and PBS. Antigen retrieval was performed with citrate (pH 6.0). Endogenous peroxidase was blocked with 3% H2O2 in water for 20 min, and nonspecific binding was blocked with diluted normal goat serum for 60 min, and was then incubated with the primary antibody (mouse anti‐human monoclonal antibody C1q (ab71089), rabbit anti‐human polyclonal antibody factor B (ab192577), rabbit anti‐human polyclonal antibody factor P (ab186834), mouse anti‐human monoclonal antibody MBL (ab23457), mouse anti‐human monoclonal antibody factor H (ab118820), mouse anti‐human monoclonal antibody C3a (ab37230), mouse anti‐human monoclonal antibody C5a (ab11877) and rabbit anti‐human polyclonal antibody SC5b‐9(ab55811), all antibodies were purchased from Abcam) for 18 h at 4°C. According to the manufacturer's instructions, labeling was identified using the SP goat IgG kit (PV‐6000, ZSGB‐Bio, China). The chromogenic reaction solution contained 3, 3′‐diaminobenzidine (DAB) (ZLI‐9018, ZSGB‐Bio, China), and counterstaining was performed with Mayer's hematoxylin (Solarbio). Slides were viewed under an imaging fluorescence microscope (Olympus BX51; Olympus).
6
Immunohistochemical Analysis of Complement Proteins
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Orbital lesion tissue slices were dewaxed, incubated in 3% H2O2 at room temperature for 5 to 10 minutes, washed with distilled water, and soaked in PBS for 5 minutes. Tissues were blocked with 10% normal goat serum at room temperature for 10 minutes before primary antibodies (C3c, Abcam, ab204121; C1q, Abcam, ab71089) were added and incubated overnight at 4°C. Tissues were then washed three times for 5 minutes with PBS before the drop-wise addition of biotin-labeled secondary antibody and incubated at 37°C for 30 minutes. Tissues were again washed three times for 5 minutes with PBS and the slices were stained using DAB reagent, washed with water, counter-stained with hematoxylin, mounted, and imaged under a microscope (OLYMPUS CX41, JAPAN).
7
Brain Protein Extraction and Quantification
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Total protein was extracted from brain homogenates using T-PER™ Tissue Protein Extraction Reagent (Thermo Scientific) added with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein concentrations were measured using a BCA protein assay kit (Thermo Scientific). Total protein (10 ~ 40 μg) was loaded on 4–20% TGX protein gel (Bio-Rad) for electrophoresis under non-reducing or reducing conditions and transferred to PVDF membranes (Bio-Rad). The membranes were blocked with Starting Block T20 (TBS) Blocking Buffer (Thermo Scientific) for 1 h at room temperature, followed by incubation with primary antibodies at 4 °C overnight. Proteins were probed with primary antibodies for IBA1 (polyclonal, cat. 016-20001, 1:400, Wako), GFAP (polyclonal, cat. ab7260, 1:500, Abcam), C1q (monoclonal, cat. ab71089, 1:1000, Abcam), and C4 (monoclonal, cat. NB200-541, 1:10, Novus Biologicals). Bands were visualized using enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific). Protein values were normalized for corresponding values of β-actin. Relative optical density was determined using ImageLab software (Bio-Rad).
8
Immunostaining of Adipose Tissue Sections
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Deparaffinized adipose tissue sections and blood vessels were fixed in 4% paraformaldehyde in phosphate-buffered saline. The sections of fat tissues and vessels were stained with hematoxylin–eosin. Immunostaining was performed overnight at 4°C with mouse anti-C1q (ab71089, Abcam,) or with rabbit anti-APN (NB100-65810, Novus Biologicals, Littleton, CO, USA) antibodies. The sections were then washed and incubated in Alexa Fluor 488 goat anti-rabbit IgG (Moleaulr Probes, Eugene, OR) and Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes, Eugene, OR) as the secondary antibody for 1 h at 37°C. The slides were examined under a Zeiss Axio Imager A1 fluorescent microscope (Carl Zeiss, Cambridge, UK).
9
Renal Pathology Assessment in Lupus Mice
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At age 20 weeks, all mice were sacrificed to collect kidneys after euthanasia by intraperitoneal injection of pentobarbital (200 mg/kg). The kidneys were formalin-fixed and paraffin-embedded and sliced at 4 μm thickness for HE staining. Immunohistochemistry of C3d, membrane attack complex (MAC) and C1q staining were conducted using antibody against C3d (R&D biosystems; AF2655), C5b-9 (Abcam; ab55811), and C1q (Abcam; ab71089). In addition, Fluorescent-dye conjugated antibodies against mouse IgG (Invitrogen; A11029) and mouse IgM mu chain (Abcam; ab150121) were used for immunofluorescence detection of IgG and IgM deposition in the kidney. Renal pathology indicators were scored by two experienced renal pathologists (HL and GL) independently based on the average score according to the activity index of renal tissue in lupus nephropathy [17 (link)]. The intensity of immunostaining was reported by GL and blindedly assessed by HL.
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