Cells were cultured in 8-well chamber slides and irradiated by LINAC at 5, 15 and 25 Gy doses. Cells were fixed 24, 72 or 120 h after radiation with 4% paraformaldehyde prior to blocking and incubation with anti-ICAM-1 antibody (ab119871, Abcam) or IgG2b isotype (ab18541, Abcam) that was fluorescently tagged using Mix-n-StainTMCFTM555 antibody labelling kit (Sigma-Aldrich), or anti-VCAM-1 antibody (ab61993, Abcam) or IgG1 isotype (ab18407, Abcam) labelled using Mix-n-StainTMCFTM640 antibody labelling kit (Sigma-Aldrich). Nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) (1 μg/mL, Life Technologies). Images were captured with fixed parameters using a confocal microscope (SP5, Leica Microsystems, Wetzlar, Germany) and analyzed with ImageJ.
Ab119871
Manufactured by Abcam
Sourced in United States
Ab119871 is a high-quality primary antibody product from Abcam. It is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab134047, by Abcam (5 mentions)
Ab18407, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ab115700, by Abcam (1 mentions)
Fjk 16, by Thermo Fisher Scientific (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Ab23875, by Abcam (1 mentions)
Ab205, by Abcam (1 mentions)
Ab56788, by Cell Signaling Technology (1 mentions)
Ab193216, by Abcam (1 mentions)
Ab131512, by Abcam (1 mentions)
Ab57602, by Cell Signaling Technology (1 mentions)
Ab71498, by Abcam (1 mentions)
Ab56889, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence substrate, by Applygen (1 mentions)
Ab8295, by Abcam (1 mentions)
Ab133504, by Abcam (1 mentions)
Ab111101, by Abcam (1 mentions)
Ab78547, by Abcam (1 mentions)
11 protocols using ab119871
1
Radiation-Induced ICAM-1 and VCAM-1 Expression
2
Histological Analysis of Tissue Samples
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Samples of equal size were taken from the ear skin, lymph nodes, and small intestine and fixed in 10% neutral buffered formalin for 24 h at 4°C. Paraffin-embedded tissue sections (3–4-μm thick) were stained with hematoxylin and eosin. The sections were stained with toluidine blue for detection of mast cells in the skin and periodic acid-Schiff (PAS) for detection of goblet cells in the small intestine. Intercellular adhesion molecule-1 (ICAM-1; ab119871, Abcam, Cambridge, MA, United States) and thymic stromal lymphopoietin (TSLP; ab115700, Abcam) in the epidermis and dermis of skin, Foxp3 (FJK-16s, Thermo Fisher) in lymph nodes, and tight junction markers, such as claudin-5 (4C3C2, Thermo Fisher), occludin (OC-3F10, Thermo Fisher), and zonula occludens (ZO)-1 (61–7,300, Thermo Fisher), in the tissues of the small intestine were observed by avidin-biotin-peroxidase complex-based immunohistochemical staining. The histological profiles of individual cross-sections of ear skin were observed under a light microscope (Eclipse 80i, Nikon, Tokyo, Japan).
3
Western Blot Analysis of Mitochondrial Dynamics Proteins
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Proteins (60–80 µg) were loaded on a 10–15% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, proteins were transferred to a PVDF membrane. Membrane were blocked with 5% nonfat dried milk (in TBST) for 2 h at room temperature and then incubated overnight at 4 °C with primary antibodies. The membrane was subsequently washed with TBST (5 min × 3) and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 1 h at room temperature [33] (link). After washing with TBST (5 min × 3), bands were detected by enhanced chemiluminescence substrate (Applygen). The antibodies used in the present were as follows: Drp1 (1:1000, Abcam, #ab56788), p-Drp1 (Ser616) (1:1000, Cell Signaling Technology, #3455), p-Drp1 (Ser637) (1:1000, Abcam, #ab193216), Fis1 (1:1000, Abcam, #ab71498), Mfn2 (1:1000, Abcam, #ab56889), Mfn1 (1:1000, Abcam, #ab57602), Mff (1:1000, Cell Signaling Technology, #86668), F-actin (1:1000, Abcam, #ab205), G-actin (1:1000, Abcam, #ab200046), p-eNOS (Ser1117) (1:1000, Abcam, #ab184154), ICAM1 (1:1000, Abcam, #ab119871), VCAM1 (1:1000, Abcam, #ab134047), AMPK (1:1000, Abcam, #ab131512), p-AMPK (1:1000, Abcam, #ab23875) [34] (link).
4
Immunostaining of Cellular Markers
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The samples were first washed with cold PBS and then permeabilized in 0.1% Triton X-100 for 10 min at 4 °C. Then, 10% goat serum albumin (Invitrogen) was used to block the samples for 1 h at room temperature. The samples were incubated with primary antibodies overnight at 4 °C [29 (link)]. After three rinses in PBS, secondary antibodies were added to the samples for 1 h at room temperature [30 (link)]. The primary antibodies were: mitochondrial import receptor subunit TOM20 homolog (Abcam; cat. no. ab78547), F4/80 (1:1000, Abcam, #ab111101), troponin T (1:1000, Abcam, #ab8295), ICAM1 (1:1000, Abcam, #ab119871) and cyt-c (1:1000, Abcam, #ab ab133504). Images were observed with an inverted microscope (magnification, 40×; BX51; Olympus Corp.).
5
Western Blot Analysis of Cardiac Proteins
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Cardiac tissue samples for western blotting were obtained from the infarcted area. Proteins isolated from sI/R-treated CMECs were also analyzed through western blots. In brief, proteins were separated by SDS electrophoresis and transferred to membranes using standard protocols,67 (link) after which they were probed with antibodies against p-eNOS (Ser1117) (1:1,000, #ab184154; Abcam), ET-1 (1:1,000, #ab2786; Abcam), ICAM1 (1:1,000, #ab119871; Abcam), VCAM1 (1:1,000, #ab134047; Abcam), Gr1 (1:1,000, #ab25377; Abcam), FUNDC1 (1:1,000, #ab224722; Abcam), ATG5 (1:1,000, #12994, Cell Signaling Technology), and Beclin1 (1:1,000, #3738; Cell Signaling Technology). The blots were visualized by chemiluminescence (ECL, Immunobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, MA, USA), and the signals were quantified by densitometry. GAPDH (analyzed with an antibody from Santa Cruz Biotechnology) served as a loading control.68 (link)
6
Neuroinflammation Biomarkers Immunodetection
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Immunohistochemistry was performed as described previously.17 The primary antibodies used were as follows: TNF‐α (ab183218; Abcam), IL‐6 (ab214429; Abcam), IL‐1β (sc‐52012; Santa Cruz), Iba‐1 (sc‐32725; Santa Cruz), ICAM‐1 (ab119871; Abcam), NOX1 (bs‐3682R; Bioss), and NOX4 (bs‐1091R; Bioss). The images were analyzed according to the immunoreactivity intensity (brown) using the Image J software. All procedures were carried out in a double‐blinded manner.
7
Immunohistochemical Analysis of Aortic Markers
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Immunohistochemical staining of the aortas was performed to detect the expression of HSP22, PPAR-γ and intracellular adhesion molecule 1 (ICAM-1) as previously described.21 (link) Briefly, the aortas were embedded in paraffin, and 5-μm sections were cut from the embedded blocks. After heat-induced antigen retrieval, the slides were incubated at 4°C overnight with the following primary antibodies: HSP22 antibody (Abcam, ab151552, rabbit monoclonal, at 1:50, USA), PPAR-γ antibody (CST, 2435, rabbit polyclonal, at 1:200, USA) or ICAM-1 antibody (Abcam, ab119871, rat monoclonal, at 1:200, USA). The slides were then incubated with biotinylated IgG (1:250) for 1 h followed by incubation with streptavidin-HRP for 30minat room temperature. DAB was added to each slide for 5 min. The slides were observed under a microscope (Olympus, Japan), and images were analyzed using Image-Pro Plus 6.0 System (Media Cybernetics, USA).41 (link)
8
Western Blot Analysis of Oxidative Stress Markers
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Total proteins were separated by SDS-PAGE, transferred on to PVDF membranes, and incubated with the following primary antibodies: anti-iNOS (1 : 1000, ab178945, Abcam), anti-p65NF-Κb (1 : 1000, 8242S, Cell Signaling Technology), anti-IκBα (1 : 1000, 4812S, Cell Signaling Technology), anti-p-IκBα (1 : 1000, 2859S, Cell Signaling Technology), anti-Histone (1 : 1000, 4499S, Cell Signaling Technology), anti-VCAM-1(1 : 1000, ab134047, Abcam), anti-ICAM-1 (1 : 1000, ab119871, Abcam), anti-COX-2 (1 : 1000, 12282S, Cell Signaling Technology), anti-cleaved PARP (1 : 1000, 5625S, Cell Signaling Technology), anti-cleaved caspase-3 (1 : 1000, 9661S, Cell Signaling Technology), anti-Bax(1 : 1000, 2772S, Cell Signaling Technology), anti-Bcl2(1 : 1000, 3498S, Cell Signaling Technology), anti-Akt (1 : 1000, 4691S, Cell Signaling Technology), anti-phospho-Akt (p-AKT) (1 : 1000, 4060S, Cell Signaling Technology), anti-Nrf2 (1 : 1000, 12721S, Cell Signaling Technology), anti-HO-1 (1 : 1000, 70081S, Cell Signaling Technology), and anti-β-actin (1 : 5000, A1978, Sigma). After further incubation with corresponding secondary antibodies, signals were detected with a chemiluminescent reagent.
9
Immunohistochemical Analysis of Tissue Markers
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The paraffin-embedded tissue was sliced into 3 μm sections. The antigen
was recovered with citrate buffer for 20 min, followed by deactivated
endogenous peroxidase with 3% H2O2 for 20 min
and sealing with 5% bovine serum albumin for 10 min. The sections were
then treated overnight at 4°C with anti-PCNA monoclonal antibody
(13110, Cell Signaling, 1:50 dilution), anti-α-SMA antibody (ab134047,
Abcam, 1:500 dilution), anti-p38 antibody (ab119871, Abcam, 1:100
dilution), and anti-phosphorylated p38-antibody (4511, Cell Signaling,
1:100 dilution). Horseradish peroxidase conjugated secondary antibody
was added to the slices after wash with phosphate-buffered saline
(PBS). The diaminobenzidine tetrahydrochloride (DAB) was used to
visualize. All images were acquired by an Olympus-IX73 microscope
(Olympus, Tokyo, Japan). At least three sections per graft were
analyzed for all markers.
was recovered with citrate buffer for 20 min, followed by deactivated
endogenous peroxidase with 3% H2O2 for 20 min
and sealing with 5% bovine serum albumin for 10 min. The sections were
then treated overnight at 4°C with anti-PCNA monoclonal antibody
(13110, Cell Signaling, 1:50 dilution), anti-α-SMA antibody (ab134047,
Abcam, 1:500 dilution), anti-p38 antibody (ab119871, Abcam, 1:100
dilution), and anti-phosphorylated p38-antibody (4511, Cell Signaling,
1:100 dilution). Horseradish peroxidase conjugated secondary antibody
was added to the slices after wash with phosphate-buffered saline
(PBS). The diaminobenzidine tetrahydrochloride (DAB) was used to
visualize. All images were acquired by an Olympus-IX73 microscope
(Olympus, Tokyo, Japan). At least three sections per graft were
analyzed for all markers.
10
Quantifying Vascular Inflammation Markers
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Anti-VCAM1: ab134047 (Abcam), anti-ICAM1: ab119871 (Abcam), anti-CD68: ab125212 (Abcam), anti-rabbit IgG: BA-1000 (Vector Laboratories), anti rat-IgG: BA-9401 (Vector Laboratories).
For quantifitative analyses, the percentage of positively stained endothelial cells (ICAM-1- and VCAM-1-stainings) or the percentage of positively stained intimal area (CD68 staining) was calculated. Of note, endothelial cells were defined based on characteristic histomorphological features (cells with thin elongated nuclei constituting the luminal lining of the vessel). A minimum of three cross sections from the proximal, medial and distal part of the vessel were analysed and a vessel mean calculated.
For quantifitative analyses, the percentage of positively stained endothelial cells (ICAM-1- and VCAM-1-stainings) or the percentage of positively stained intimal area (CD68 staining) was calculated. Of note, endothelial cells were defined based on characteristic histomorphological features (cells with thin elongated nuclei constituting the luminal lining of the vessel). A minimum of three cross sections from the proximal, medial and distal part of the vessel were analysed and a vessel mean calculated.
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