The aorta and heart were isolated from each rabbit. Aortic trees were opened and fixed in 10 % buffered formalin. After formalin fixation, the whole aortas were stained with Sudan IV and photographed for evaluation of the gross size of atherosclerotic lesions [14 (link)]. The surface sudanophilic area of each aorta segment was measured using an image analysis system. For histological analysis, serial sections (3 μm thick) were stained with H&E, Elastica van Gieson (EVG) and immunohistochemically stained with RAM11 (working dilution 400X) and HHF35 (200X) monoclonal antibodies against macrophages and smooth muscle α-actin (Dako) [14 (link)]. The microscopic lesion area, macrophages and SMCs in the lesions were quantified and early-stage lesions and advanced lesions were classified and analyzed using an image analysis system [14 (link)]. Advanced lesions refer to those which to contain a typical fibrotic cap and a lipid or necrotic core with or without calcification [14 (link)]. Rabbit hearts were sectioned and coronary atherosclerosis was analyzed and expressed as stenosis %, as described previously [15 (link)].
α actin
Manufactured by Agilent Technologies
Sourced in Canada
α-actin is a structural protein found in the cytoskeleton of eukaryotic cells. It is a key component of the actin filaments, which are responsible for various cellular processes such as cell motility, cell shape maintenance, and muscle contraction.
Automatically generated - may contain errors
Lab products found in correlation
Foxo4, by Cell Signaling Technology (2 mentions)
Procaspase 3, by Cell Signaling Technology (2 mentions)
Foxo1, by Santa Cruz Biotechnology (2 mentions)
Lamp2, by Cell Signaling Technology (2 mentions)
Pico tag reverse phase column, by Waters Corporation (2 mentions)
Ampkα, by Cell Signaling Technology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ram11, by Agilent Technologies (1 mentions)
Hhf35, by Agilent Technologies (1 mentions)
Mouse and rabbit specific hrp dab abc detection ihc kit, by Abcam (1 mentions)
Ndrg2, by Santa Cruz Biotechnology (1 mentions)
Mac 1, by Roche (1 mentions)
Axioplan 2 imaging microscope, by Zeiss (1 mentions)
Axiocam axiovision, by Zeiss (1 mentions)
6 protocols using α actin
1
Quantifying Atherosclerotic Lesions in Rabbit Aortas
2
Histological Analysis of Calcified Aortic Valve
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Three consecutive sections from the AS valve analyzed by MALDI-IMS were used for the histological analysis. These sections were stained using Verhoeff-Van Giesson staining protocol for elastic fibers and oil red for lipids.
Besides the consecutive sections, we used 4 different AS valves, selecting 4 sections with increasing level of calcification of each one, for IHC analyses. In this case, sections were blocked with 10% BSA in PBS Buffer with 0.1% Tween 20 and incubated for 1 h with the primary antibodies at room temperature. Specifically, these analyses were performed with antibodies against CD68, α-actin (DAKO), NDRG2 and collagen α3 Type VI (Santa Cruz). Secondary antibody conjugated with biotin, streptavidin-peroxidase and DAB was used for all immunostainings (Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit, Abcam). Besides, additionally sections from 4 stenotic valves were used to study the distribution of NDRG2 and collagen α3 Type VI along the different stages of the lesion. For an impartial analysis of the DAB staining, an orthonormal transformation of the RGB images by using an ImageJ plugin based on Ruifrok and Johnston’s method for color deconvolution was performed37 (link).
Besides the consecutive sections, we used 4 different AS valves, selecting 4 sections with increasing level of calcification of each one, for IHC analyses. In this case, sections were blocked with 10% BSA in PBS Buffer with 0.1% Tween 20 and incubated for 1 h with the primary antibodies at room temperature. Specifically, these analyses were performed with antibodies against CD68, α-actin (DAKO), NDRG2 and collagen α3 Type VI (Santa Cruz). Secondary antibody conjugated with biotin, streptavidin-peroxidase and DAB was used for all immunostainings (Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit, Abcam). Besides, additionally sections from 4 stenotic valves were used to study the distribution of NDRG2 and collagen α3 Type VI along the different stages of the lesion. For an impartial analysis of the DAB staining, an orthonormal transformation of the RGB images by using an ImageJ plugin based on Ruifrok and Johnston’s method for color deconvolution was performed37 (link).
3
Immunohistological Analysis of Tissue Samples
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Immunohistology was performed as previously described [24 (link), 27 (link)]. Immunoreactions were achieved with monoclonal antibodies (mAbs) directed against α-actin (DAKO, Glostrup, Denmark), superoxide dismutase 2 (SOD2), or integrin subunit alpha M (Mac-1, Roche, Mannheim, Germany). Mouse anti-human major histocompatibility complex, class II, DR Alpha (HLA-DRA, LN3 clone, Roche Mannheim, Germany) and mouse anti- tumor protein P53 (Tp53, DAKO), overnight at RT. After washing in phosphate-buffered saline (PBS), the sections were incubated with biotinylated anti-mouse IgG streptavidin-peroxidase (HRP) conjugated (Amersham, Braunschweig, Germany); afterward staining reaction was performed by adding a diaminobenzidine (DAB) solution (Pierce, Rockford, IL, USA), and stained with Mayer’s hematoxylin. From the histological and immunohistological sections, digitalized images were obtained using an Axioplan2 imaging microscope (Carl Zeiss GmbH, Jena, Germany) and the digital high-resolution imaging system AxioCam/AxioVision (Carl Zeiss GmbH).
4
Measuring Muscle Amino Acid Metabolism
5
Measuring Muscle Amino Acid Metabolism
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Immediately following euthanasia, LD samples were stored at −70°F until processing. Free intracellular AA concentrations in muscle were determined by high-performance liquid chromatography (HPLC; PICO-TAG reverse-phase column; Waters, Mildford, MA,) using an analytical method based on deproteinization and derivatization of AA with phenylisothiocyanate, as previously described (18 (link)).
Amino acid transporter and intracellular markers of protein degradation and autophagy were measured by immunoblotting analysis, as previously described (8 (link), 31 (link)). The antibodies used in the immunoblotting process were PKB (total and Ser473, Cell Signaling Technology, Beverly, MA), AMPK-α (total and Thr172, Cell Signaling Technology, Beverly, MA), FOXO1 (total protein, Santa Cruz Technology, Santa Cruz, CA; Ser256, Cell Signaling Technology), FOXO4 (total protein and Ser262, Cell Signaling Technology, Beverly, MA), pro-caspase 3 (total protein, Cell Signaling Technology, Beverly, MA), MuRF1 (ECM Biosciences, Versailles, KY), atrogin-1 (ECM Biosciences, Versailles, KY), LAT1 (Cell Signaling Technology, Beverly, MA), SNAT2 (Aviva System Biology, San Diego, CA.), LC3 (Cell Signaling Technology, Beverly, MA), β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), α-Actin (40 KDa; Dako-Cytomation, Glostrup, Denmark) and LAMP-2 (Cell signaling Technology, Danvers, MA)
Amino acid transporter and intracellular markers of protein degradation and autophagy were measured by immunoblotting analysis, as previously described (8 (link), 31 (link)). The antibodies used in the immunoblotting process were PKB (total and Ser473, Cell Signaling Technology, Beverly, MA), AMPK-α (total and Thr172, Cell Signaling Technology, Beverly, MA), FOXO1 (total protein, Santa Cruz Technology, Santa Cruz, CA; Ser256, Cell Signaling Technology), FOXO4 (total protein and Ser262, Cell Signaling Technology, Beverly, MA), pro-caspase 3 (total protein, Cell Signaling Technology, Beverly, MA), MuRF1 (ECM Biosciences, Versailles, KY), atrogin-1 (ECM Biosciences, Versailles, KY), LAT1 (Cell Signaling Technology, Beverly, MA), SNAT2 (Aviva System Biology, San Diego, CA.), LC3 (Cell Signaling Technology, Beverly, MA), β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), α-Actin (40 KDa; Dako-Cytomation, Glostrup, Denmark) and LAMP-2 (Cell signaling Technology, Danvers, MA)
6
Quantification of CYP4A1 and CYP4F3 in Aorta
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Frozen thoracic aorta was homogenized in RIPA lysis buffer (Thermo Fisher Scientific) [50 mmol/L of Tris/HCl (pH 7.4), 1% Igepal, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 2 μg/ml protease inhibitor] and centrifuged at 14000 g for 30 min at 4°C. Equal amount of protein (30 μg) from each aorta was resolved by SDS-PAGE on 4–12% gels and electroblotted onto nitrocellulose membrane. Membranes were incubated overnight at 4°C with 1:1000 dilution of polyclonal CYP4A1 and CYP4F3 antibodies (Biorbyt, Cambridge, United Kingdom). After that, membranes were incubated for 1 h with a 1:2000 dilution of horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Thermo Scientific, Waltham, MA, United States) and chemiluminescent signal was visualized by ImageQuant LAS 4000 imaging system (GE Headquarter Life Science, Marlborough, MA, United States). Densitometric analyses of immunoblots were performed using an imaging software (Image J, NIH, United States), and CYP4A1 and CYP4F3 band densities were normalized by densitometry of α-actin (1:2000 – Dako).
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