Mice were sacrificed 14 d after FAL to collect ischaemic left gastrocnemius muscle. 7-μm thick cryosections were first blocked with 5% normal goat serum and then incubated with primary antibodies CD31 (a marker for endothelial cell, Santa Cruz, CA) and α-smooth muscle actin (α-SMA) (Abcam, UK, Cambridge) at 4 °C overnight, followed by the incubation with secondary antibodies Alexa-488 conjugated anti-rabbit IgG and Alexa-594 conjugated anti-mouse (1:500; Jackson ImmunoResearch Laboratories, PA, USA) for 1 h at room temperature. Subsequently, sections were mounted using Vectashield with DAPI (4'6-diamino-2-fenilindol dihidrocloreto) for counter-staining of nuclei before observation. Pictures from each section were taken under 400× magnification, using Olympus BX51 high-magnification microscope. Capillaries were identified by positive staining for CD31 and those displaying a second cellular layer stained with SMA, surrounding the inner one, were counted as arterioles. According to the procedures previously published [25 (link),26 (link)], ten different fields from each tissue section were selected randomly. Capillaries labelled with CD31 were counted. Capillary density was expressed as the number of capillaries per square millimetre. The proportion of fibres with central nucleus (regenerated fibres) in the injured area was calculated [27 (link)].
Alexa488 conjugated anti rabbit igg
Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom
Alexa488-conjugated anti-rabbit IgG is a secondary antibody labeled with the Alexa Fluor 488 fluorescent dye. It is designed to detect and bind to rabbit immunoglobulin G (IgG) proteins in various immunoassay and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 high magnification microscope, by Olympus (2 mentions)
α smooth muscle actin α sma, by Abcam (2 mentions)
Anti nrf2, by Cell Signaling Technology (2 mentions)
Anti g9a, by Cell Signaling Technology (2 mentions)
Hrp conjugated anti rabbit total igg, by Jackson ImmunoResearch (2 mentions)
Pe conjugated f4 80, by Cytek Biosciences (2 mentions)
Anti h3k9me1, by Cell Signaling Technology (2 mentions)
Anti ser9 phospho gsk 3β, by Cell Signaling Technology (2 mentions)
Anti ho 1, by Cell Signaling Technology (2 mentions)
Anti trxr1, by Cell Signaling Technology (2 mentions)
Anti lrp2 antibody, by Santa Cruz Biotechnology (2 mentions)
Anti srebp2 antibody, by Abcam (2 mentions)
Anti phospho β catenin ser33 37 thr41, by Cell Signaling Technology (2 mentions)
Filipin, by Merck Group (2 mentions)
Anti β catenin, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (2 mentions)
Anti h3k9me2, by Cell Signaling Technology (2 mentions)
Anti sirt6, by Cell Signaling Technology (2 mentions)
Hrp conjugated anti β actin antibody, by Merck Group (2 mentions)
Anti h3k9ac, by Cell Signaling Technology (2 mentions)
10 protocols using alexa488 conjugated anti rabbit igg
1
Quantifying Muscle Angiogenesis in Ischemic Mice
2
Immunofluorescence Analysis of Cellular Markers
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HRP-conjugated anti-β-ACTIN antibody, Filipin and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich/ Merck Millipore. Alexa488-conjugated anti-rabbit IgG, HRP-conjugated anti-rabbit total IgG and light chain specific IgG antibodies were purchased from Jackson ImmunoResearch, USA; PE-conjugated F4/80 was procured from Tonbo Biosciences, USA. Alexa Fluor 660 conjugated CD68 was purchased from ThermoFischer Scientific. Anti-G9a, anti-SIRT6, anti-H3K9me1, anti-H3K9me2, anti-H3K9Ac, anti-Ser33/37/Thr41 phospho-β-CATENIN, anti-Ser9 phospho-GSK-3β, anti-β-CATENIN, anti-NRF2, anti-HO1 and anti-TRXR1 antibodies were obtained from Cell Signaling Technology, USA. Anti-LRP2 antibody was purchased from Santa Cruz Biotechnology, USA; anti-SREBP2 antibody was procured from Abcam, USA; and anti-NQO1 antibody was purchased from Calbiochem, USA.
3
Quantifying Amyloid-β Burden and Smooth Muscle Cell Fragmentation in Pial Vessels
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To quantify CAA burden and Aβ-associated fragmentation of smooth muscle cells in pial vessels [20 (link), 31 (link)], tangential brain sections were incubated with anti-Aβ (4G8, 1:1,000, mouse; Covance) and the smooth muscle marker anti-α-actin (1:300, rabbit, Abcam) for 48 h. After washing, sections were labeled with Alexa 488-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch) and Alexa 647-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch). Pial arterioles (n = 30–50/group) positive for Aβ and α-actin, ranging in diameter from 20 to 100 μm, were randomly imaged by confocal microscope (63x). Aβ accumulation around pial-penetrating arteries was assessed by the ratio (%) between 4G8+ Aβ immunoreactivity and α-actin+ pial vessel area obtained with ImageJ in the same sections in which smooth muscle fragmentation was quantified. However, the A β specie (1–40, 1–42) was not determined. Smooth muscle cell fragmentation was quantified by counting α-actin fragments in pial-penetrating arteriole using ImageJ, expressed as the fragmentation index: 100 − [(1/number of α-actin fragments) × 100].
4
Immunofluorescence and Immunoenzyme Staining of SLC7a10 in Mouse Brain
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After perfusion with 4% paraformaldehyde (PFA) in phosphate buffer, brains were removed and post-fixed in 4% PFA overnight (~ 12 h) at 4 °C. All brains were washed in PBS solution including 30% sucrose and snap-frozen; frozen sections of 30-µm thickness were then cut. All histological procedures were performed as described previously [3 (link)].
For primary antibody, anti-SLC7a10 (N-terminal) (1:200, rabbit polyclonal, GTX47874, GeneTex, USA) was used. Alexa 488-conjugated anti-rabbit IgG (1:1000, Jackson ImmunoResearch Laboratories, UK) was used for secondary antibody in immunofluorescence staining. Counterstaining was performed by blue-fluorescent Nissl stain (1:200, Neurotrace 435/455, Thermo Fisher Scientific). Fluorescence images were obtained with a confocal laser scanning microscope (Nikon C2-NiE, Japan). To obtain 3D deconvolution images, we used a confocal laser scanning microscope (Olympus FV3000, Japan). For immunoenzyme staining, we used anti-rabbit IgG combined with amino acid polymers and peroxidase (Histofine Simple Stain MAX PO Kit, 414141F, Nichirei Bioscience, Japan) for secondary antibody. The peroxidase color reaction was performed in diaminobenzidine tetrahydrochloride (DAB) solution (DAB Substrate Kit, SK-4100, Vector Laboratories, USA).
For primary antibody, anti-SLC7a10 (N-terminal) (1:200, rabbit polyclonal, GTX47874, GeneTex, USA) was used. Alexa 488-conjugated anti-rabbit IgG (1:1000, Jackson ImmunoResearch Laboratories, UK) was used for secondary antibody in immunofluorescence staining. Counterstaining was performed by blue-fluorescent Nissl stain (1:200, Neurotrace 435/455, Thermo Fisher Scientific). Fluorescence images were obtained with a confocal laser scanning microscope (Nikon C2-NiE, Japan). To obtain 3D deconvolution images, we used a confocal laser scanning microscope (Olympus FV3000, Japan). For immunoenzyme staining, we used anti-rabbit IgG combined with amino acid polymers and peroxidase (Histofine Simple Stain MAX PO Kit, 414141F, Nichirei Bioscience, Japan) for secondary antibody. The peroxidase color reaction was performed in diaminobenzidine tetrahydrochloride (DAB) solution (DAB Substrate Kit, SK-4100, Vector Laboratories, USA).
5
Immunostaining of Mouse Brain Slices
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To immunostain brain slices, adult mouse brains were infused with Tissue-Tek (Sakura Finetek, Tokyo, Japan), frozen in liquid nitrogen and sectioned at 10 µm using a cryostat (HM500-OM, Carl Zeiss, Oberkochen, Germany). The sections were mounted on silane-coated coverslips and dried for ~30 min at room temperature. The samples were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, and 2.7 mM KCl, pH 7.4) for 10 min at room temperature and permeabilized with 0.5% Triton X-100 in PBS. After blocking with 10% FCS in PBS, the samples were incubated with the anti-RNG105 antibody over night at 4°C. After washing with PBS, the samples were incubated with Alexa488-conjugated anti-rabbit IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA) and 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) (Wako Pure Chemical Industries) for 1 hr at room temperature to label RNG105 and nuclei, respectively. To immunostain cultured neurons, neurons at 12 DIV were fixed and stained in the same way. Fluorescence images were acquired using an A1 confocal laser microscope equipped with a Ti-E inverted microscope (Nikon, Tokyo, Japan) with a 10 × objective lens or a PlanApo VC60 × oil objective lens.
6
Quantifying Muscle Angiogenesis in Ischemic Mice
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Mice were sacrificed 14 d after FAL to collect ischaemic left gastrocnemius muscle. 7-μm thick cryosections were first blocked with 5% normal goat serum and then incubated with primary antibodies CD31 (a marker for endothelial cell, Santa Cruz, CA) and α-smooth muscle actin (α-SMA) (Abcam, UK, Cambridge) at 4 °C overnight, followed by the incubation with secondary antibodies Alexa-488 conjugated anti-rabbit IgG and Alexa-594 conjugated anti-mouse (1:500; Jackson ImmunoResearch Laboratories, PA, USA) for 1 h at room temperature. Subsequently, sections were mounted using Vectashield with DAPI (4'6-diamino-2-fenilindol dihidrocloreto) for counter-staining of nuclei before observation. Pictures from each section were taken under 400× magnification, using Olympus BX51 high-magnification microscope. Capillaries were identified by positive staining for CD31 and those displaying a second cellular layer stained with SMA, surrounding the inner one, were counted as arterioles. According to the procedures previously published [25 (link),26 (link)], ten different fields from each tissue section were selected randomly. Capillaries labelled with CD31 were counted. Capillary density was expressed as the number of capillaries per square millimetre. The proportion of fibres with central nucleus (regenerated fibres) in the injured area was calculated [27 (link)].
7
Immunoblot Analysis of Angiogenic Factors
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Primary antibodies for immunoblot analyses were purchased from commercial
sources, including actin (horseradish peroxidase (HRP); I-19), nitric oxide
synthase (NOS) 3 (C-20), VEGF (147) antibodies from Santa Cruz Biotechnology
Inc. (Santa Cruz, CA, USA); p-eNOS (ser1177) antibodies from Cell Signaling
(Danvers, MA, USA); anti-smooth muscle α-actin, mouse anti-3nitrotyrosine (39B6)
and mouse anti-glutathione (D8) from Abcam (Cambridge, MA, USA); CD31 from BD
Bioscience (San Jose, CA, USA); rabbit anti-3nitrotyrosine from Invitrogen
(Waltham, MA, USA); rabbit and mouse peroxidase-conjugated secondary antibody
also from Cell Signaling. Secondary antibodies Alexa-594–conjugated anti-rat
IgG, Alexa-488–conjugated anti-rabbit IgG, Rhodamine Red-X anti-rabbit IgG and
Fluorescein FITC anti-mouse IgG were purchased from Jackson ImmunoResearch
Laboratories (West Grove, PA, USA). CGP 42112A was purchased from Bachem
(Torrance, CA, USA), GKT136901 from Millipore (Etobicoke, ON, USA) and
N-acetyl-cystein from Sigma (St. Louis, MO, USA).
sources, including actin (horseradish peroxidase (HRP); I-19), nitric oxide
synthase (NOS) 3 (C-20), VEGF (147) antibodies from Santa Cruz Biotechnology
Inc. (Santa Cruz, CA, USA); p-eNOS (ser1177) antibodies from Cell Signaling
(Danvers, MA, USA); anti-smooth muscle α-actin, mouse anti-3nitrotyrosine (39B6)
and mouse anti-glutathione (D8) from Abcam (Cambridge, MA, USA); CD31 from BD
Bioscience (San Jose, CA, USA); rabbit anti-3nitrotyrosine from Invitrogen
(Waltham, MA, USA); rabbit and mouse peroxidase-conjugated secondary antibody
also from Cell Signaling. Secondary antibodies Alexa-594–conjugated anti-rat
IgG, Alexa-488–conjugated anti-rabbit IgG, Rhodamine Red-X anti-rabbit IgG and
Fluorescein FITC anti-mouse IgG were purchased from Jackson ImmunoResearch
Laboratories (West Grove, PA, USA). CGP 42112A was purchased from Bachem
(Torrance, CA, USA), GKT136901 from Millipore (Etobicoke, ON, USA) and
N-acetyl-cystein from Sigma (St. Louis, MO, USA).
8
Antibody Usage for Immunofluorescence and Immunoblotting
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Antibodies used for immunofluorescence (IF) microscopy were mouse anti-FLAG (M2, Sigma-Aldrich Cat. No. F3165), and rabbit anti-PML H238 (Santa Cruz Biotechnology Cat. No. sc5621); Cy5-, Alexa647-, or Alex488-conjugated anti-mouse IgG (Jackson ImmunoResearch Cat. No. 715–175–151, 715–605–151, 715–545–151); Cy3-, Cy5-, FITC-, or Alexa488-conjugated anti-rabbit IgG (Jackson ImmunoResearch Cat. No. 711–165–152, 711–175–152, 711–095–152, 711–545–152). All antibodies for IF microscopy were diluted 1:100. Antibodies used for immunoblotting were mouse anti-FLAG HRP (M2, Sigma-Aldrich Cat. No. A8592); mouse anti-histone H3 1B1B2 (Cell Signaling Technology Cat. No 2367S), rabbit anti-HA C29F4 (Cell Signaling Technology Cat. No. 3724S), anti-rabbit IgG-HRP (Cell Signaling Technology Cat. No. 7074S); anti-mouse IgG-HRP (GE Healthcare Cat. No NA931V). Primary antibodies for immunoblotting were diluted 1:1000–1:2500; HRP-conjugated antibodies were diluted 1:5000–1:10,000.
9
Quantification of Aβ-Induced Smooth Muscle Fragmentation
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To quantify Aβ-associated fragmentation of smooth muscle cells in pial vessels [20 (link), 30 (link)], brain sections were incubated with anti-Aβ (4G8, 1:1,000, mouse; Covance) and the smooth muscle marker anti-α-actin (1:300, rabbit, Abcam) for 48 hr. After washing, sections were labeled with Alexa 488-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch) and Alexa 647-conjugated anti-rabbit IgG (1:200; Jackson ImmunoResearch). Pial arterioles (n = 30–50/group) positive for Aβ and α-actin, ranging in diameter from 20 to 100 µm, were randomly imaged by confocal microscope (63x). The fragmentation of smooth muscles was quantified by counting the number of α-actin fragments of each arteriole using ImageJ, expressed as the fragmentation index: 100 − [(1/number of α-actin fragments) x 100].
10
Immunostaining for Cell Signaling
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HRP-conjugated anti-β-ACTIN antibody, Filipin and 4′,6-Diamidino-2phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich/ Merck Millipore. Alexa488-conjugated anti-rabbit IgG, HRP-conjugated anti-rabbit total IgG and light chain specific IgG antibodies were purchased from Jackson ImmunoResearch, USA; PE-conjugated F4/80 was procured from Tonbo Biosciences, USA. Anti-G9a, anti-SIRT6, anti-H3K9me1, anti-H3K9me2, anti-H3K9Ac, anti-Ser33/37/Thr41 phospho-β-CATENIN, anti-Ser9 phospho-GSK-3β, anti-β-CATENIN, anti-NRF2, anti-HO1 and anti-TRXR1 antibodies were obtained from Cell Signaling Technology, USA. Anti-LRP2 antibody was purchased from Santa Cruz Biotechnology, USA; anti-SREBP2 antibody was procured from Abcam, USA; and anti-NQO1 antibody was purchased from Calbiochem, USA.
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