Rabbit anti lox 1 antibody

Manufactured by Abcam

Sourced in United Kingdom

Rabbit anti-LOX-1 antibody is a primary antibody that specifically binds to the lectin-like oxidized LDL receptor-1 (LOX-1) protein. LOX-1 is a type II membrane protein that functions as a receptor for oxidized low-density lipoprotein (ox-LDL) and is involved in the pathogenesis of atherosclerosis.

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7 protocols using rabbit anti lox 1 antibody

Histological Analysis of Atherosclerotic Lesions

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Cardiac tissues were fixed in 10% buffered formalin for 30 min, dehydrated overnight in 75% ethanol, and embedded in paraffin. Serial sections (4 μm) were cut for morphometric analysis of the atherosclerotic lesions. The sections were stained with H&E, Masson’s trichrome, and Periodic Acid-Schiff (PAS) for histological analysis. For immunohistochemical staining, the heart sections were deparaffinized and rehydrated. Next, the sections were blocked with 3% H₂O₂ in methanol for 15 min to inactivate the endogenous peroxidases and incubated overnight at 4 ℃ with the primary antibodies: TGF-β (rabbit anti-TGF-β antibody, 1:300; Proteintech, Wuhan, China), collagen I (rabbit anti-collagen I antibody, 1:1000; Proteintech), collagen III (rabbit anti-collagen III antibody, 1:1000; Proteintech), MMP2 (rabbit anti- MMP2 antibody, 1:200; Proteintech), MMP9 (rabbit anti- MMP9 antibody, 1:300; Proteintech), LOX-1 (rabbit anti-LOX-1 antibody, 1:300; Abcam, England, CD36 (rabbit anti-CD36 antibody, 1:500; Proteintech). The sections were then incubated for 30 min at room temperature with a goat anti-rabbit HRP secondary antibody (anti-rabbit Universal Immunohistochemical Detection Kit; Proteintech). All sections were examined with an Olympus B×40 upright light microscope (Olympus, Tokyo, Japan).

Dong M., Luo Y., Lan Y., He Q., Xu L, & Pei Z. (2023). Luteolin reduces cardiac damage caused by hyperlipidemia in Sprague-Dawley rats. Heliyon, 9(6), e17613.

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Immunohistochemical Analysis of LOX-1 and CD68

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Immunohistochemistry was performed using the Histone Simple stain kits (Nichirei, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, paraffin-embedded sections were deparaffinized with xylene and then rehydrated in a descending series of ethanol concentrations. The sections were treated for 15 min with 3% H₂O₂ in methanol to inactivate endogenous peroxidases and were then incubated at room temperature for 1 h with the primary antibodies against LOX-1 (rabbit anti-LOX-1 antibody, 1:200; Abcam, England) or CD68 (rabbit anti-CD68 antibody, 1:500; Abcam, England). All sections were analyzed using an Olympus B × 40 upright light microscope (Olympus, Tokyo, Japan). For each staining, totally 3 × 7 sections (7 mice) per group were analyzed and the representative images were presented. All image analyses were done by a blinded reviewer.

Xu J., Zhu L., Liu H., Li M., Liu Y., Yang F, & Pei Z. (2018). Thymoquinone reduces cardiac damage caused by hypercholesterolemia in apolipoprotein E-deficient mice. Lipids in Health and Disease, 17, 173.

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Aortic Histology and Immunohistochemistry

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The aorta was dissected free from the surrounding connective tissue. Aorta samples were collected and fixed in 4% paraformaldehyde. Samples were embedded in paraffin and then were cut into slices using a microtome (Leica RM 2235 or Leica CM1850UV; Leica, Solms, Germany). Slices were then mounted onto glass slides and histological examinations were performed.
Immunohistochemistry was performed using the Histone Simple Stain Kit (Nichirei, Tokyo, Japan) according to the manufacturer's instructions. Briefly, paraffin-embedded sections were deparaffinized with xylene and then rehydrated in a descending series of ethanol washes. The sections were treated for 15 min with 3% H₂O₂ in methanol to inactivate endogenous peroxidases and then incubated at room temperature for 1 h with primary antibodies to collagen IV (rabbit anti-collagen IV antibody, 1:500; Abcam, England) and LOX-1 (rabbit anti-LOX-1 antibody, 1:250; Abcam). All sections were observed under an Olympus B ×40 upright light microscope (Olympus, Tokyo, Japan).

Zhang Y., Wang N., Zhu L., Liu Y., Pei Z., Wang G., Luo L, & Liu H. (2017). The Dipeptidyl Peptidase-4 Inhibitor Teneligliptin Reduces Aortic Damage from Hypercholesterolaemia in Apolipoprotein E-Deficient Mice. Biomedicine Hub, 2(2), 1-9.

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Immunohistochemistry of Kidney Samples

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Kidney samples were collected and fixed in 4% paraformaldehyde. Samples embedded in paraffin were cut into slices using a microtome (Leica RM2235 or Leica CM1850UV, Solms, Germany). The slices were then mounted onto glass slides, and immunohistochemistry was performed using the Histone Simple Stain Kit (Nichirei, Tokyo, Japan) according to the manufacturer's protocol. Briefly, paraffin-embedded sections were deparaffinized with xylene and then rehydrated with serially diluted water-ethanol solution. The sections were treated with 3% H₂O₂ in methanol to inactivate endogenous peroxidases for 15 min and incubated with primary antibodies for CD68 (rabbit anti-CD68 antibody, 1 : 500; Abcam, UK) or LOX-1 (rabbit anti-LOX-1 antibody, 1 : 250; Abcam) at room temperature for 1 h. All sections were observed with ×40 objective lenses under an upright microscope (Olympus, Tokyo, Japan).

Xu J., Pei Z., Yu M., Li X., Wang L., Lin Y., Chen X, & Liu X. (2020). Combinational Use of Antiplatelet Medication Sarpogrelate with Therapeutic Drug Rosuvastatin in Treating High-Cholesterol Diet-Induced Chronic Kidney Disease in ApoE-Deficient Mice. BioMed Research International, 2020, 1809326.

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Western Blot Analysis of Cardiac Proteins

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Proteins were extracted from heart tissues using radioimmunoprecipitation assay buffer (P0013B; Beyotime, Shanghai, China). The samples were electrophoresed on 10% SDS-PAGE gels and proteins were transferred to polyvinylidene fluoride membranes (PVDF) (Immobilon, Millipore, Billerica, MA, USA). The membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBS-T) containing 5% skim milk, then incubated in primary antibody diluents (P0023A; Beyotime), and gently shaken overnight at 4 °C. Primary antibodies against LOX-1 (rabbit anti-LOX-1 antibody, 1:250; Abcam), phospho-ERK (Rabbit anti-phospho-ERK, 1:1000; Cell Signalling Technology), and β-actin (1:1000; Cell Signalling Technology) were used. The membranes were then incubated with the secondary antibody (anti-rabbit Ig-G, 1:1000; Cell Signalling Technology) for 1 h. This analysis was independently performed three times. The protein levels are expressed as protein/β-actin ratios to minimize loading differences. The relative signal intensity was quantified using NIH Image J software.

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Western Blot Analysis of LOX-1 Protein

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Proteins were extracted from renal cortical tissues using RIPA lysis buffer (Beyotime P0013B, Shanghai, China). Samples were electrophoresed on 10% gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were then transferred to a polyvinylidene fluoride membrane (Immobilon, Millipore, MA, USA). The membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% skimmed milk, incubated in primary antibody diluents (P0023A; Beyotime), and gently shaken overnight at 4°C. Primary antibodies against LOX-1 (rabbit anti-LOX-1 antibody, 1 : 500; Abcam) and anti-β-actin (1 : 1000; Cell Signaling Technology) were added. Then, the membranes were incubated with a secondary antibody (anti-rabbit immunoglobulin G, 1 : 1000; Cell Signaling Technology) for 1 h. All these experiments were repeated independently three times. The expression levels of these proteins were normalized to the amount of β-actin proteins to minimize existing differences. The signal intensities were quantified using NIH ImageJ software.

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Quantification of LOX-1 and Erk Proteins

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Western blot analysis was performed using standard procedures50 (link). The lysates were separated on Any kD Mini-PROTEAN TGX Precast Gels (Bio-Rad), followed by immunoblotting using a primary antibody to LOX-1 (rabbit anti-LOX-1 antibody, 1:250; Abcam), total Erk1(Rabbit anti-Erk, 1:1000; Cell Signaling Technology, USA), Phospho-Erk (Rabbit anti-phospho-Erk, 1:2000; Cell Signaling Technology), anti-β-actin (1:1000; Cell Signaling Technology). Membranes were then incubated with secondary antibody (anti-rabbitIg-G, 1:1000; Cell Signaling Technology). This analysis was carried out independently three times. Protein levels are expressed as protein/β-actin ratios to minimize loading differences. The relative signal intensity was quantified using NIH Image J software.

Pei Z., Okura T., Nagao T., Enomoto D., Kukida M., Tanino A., Miyoshi K.I., Kurata M, & Higaki J. (2016). Osteopontin deficiency reduces kidney damage from hypercholesterolemia in Apolipoprotein E-deficient mice. Scientific Reports, 6, 28882.

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