Western blot analysis was utilized to evaluate the expression of IL-1β, IL-6 and ICAM-1 in mouse gingival tissues. Briefly, palatal gingival tissues were collected (2.0 mm × 2.5 mm) from mice in all the three groups 2 h after transfection. Total protein was then extracted from each tissue using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and soluble proteins were resolved via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Immobilon-P, Millipore, Darmstadt, Germany). The blots were subsequently probed with a primary antibody for IL-6 (1:1 000; Novus Biologicals, Littleton, CO, USA), IL-1β (1:1 000; Santa Cruz Biotechnology, Dallas, TX, USA) or ICAM-1 (1:1 000; Proteintech, Chicago, IL, USA). Equal protein loading was confirmed by probing for β-actin(1:2 000; Santa Cruz Biotechnology). For chemiluminescence detection, we used a peroxidase-conjugated anti-rabbit IgG secondary antibody (1:10 000; Santa Cruz Biotechnology). The band intensities were measured using a ChemiDoc image analysis system (Fuji Film, Tokyo, Japan).
Icam 1
Manufactured by Proteintech
Sourced in United States, China
ICAM-1 is a cell surface glycoprotein that functions as an intercellular adhesion molecule. It is involved in the binding of leukocytes to endothelial cells and in cell-cell interactions.
Automatically generated - may contain errors
Lab products found in correlation
Vcam 1, by Proteintech (7 mentions)
Gapdh, by Proteintech (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Itgb4, by Proteintech (2 mentions)
Hif 1α, by Proteintech (2 mentions)
Occludin, by Abcam (2 mentions)
Vcam 1, by Abcam (2 mentions)
Claudin 5, by Thermo Fisher Scientific (2 mentions)
Il 1β, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated anti rabbit igg secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Nf κb p65, by Abcam (1 mentions)
P iκbα, by Bioworld Technology (1 mentions)
P ampk, by Proteintech (1 mentions)
Dmem culture medium, by Thermo Fisher Scientific (1 mentions)
β actin, by Proteintech (1 mentions)
Tnf α, by Proteintech (1 mentions)
Vcam 1, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
29 protocols using icam 1
1
Gingival Expression of Inflammatory Markers
2
Immunohistochemical Analysis of Murine Tumors
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Preliminary process of tumor tissues from mice was similar to that of IHC. Antigen retrieval was performed by high-pressure heating with Tris-EDTA buffer (pH 9.0). Three percent H2O2 was used to block endogenous peroxidase activity. After serum blocking, the sections were then incubated overnight at 4 °C with mouse ICAM1 (Proteintech, #60299) and CD8 (Abcam, ab217344) antibodies. After washing with PBS-T, the sections were incubated with Dylight 488-conjugated goat anti-mouse IgG(H + L) (Abbkine, #A23210) and Dylight 594-conjugated goat anti-rabbit IgG(H + L) antibodies (Abbkine, #A23420) for 1 h at room temperature. Nuclei were stained with DAPI (Abcam, ab104139).
3
Western Blot Analysis of Inflammatory Markers
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Operations were carried out strictly according to kit instructions. The entire process of producing a Western blot includes sample preparation, gel electrophoresis, transfer from gel to membrane, and immunostain of the blot. The primary antibodies were added according to the manual and with the following dilutions: MCP-1 (batch number: 131120w, manufacturer: Beijing Bioss Biotechnology Co., Ltd., China) 1 : 250, ICAM-1 (batch number: 00025688, manufacturer: Proteintech Group, USA) 1 : 250, NF-κBp65 (batch number: GR194772-1, manufacturer: Abcam, England) 1 : 2000, p-IκBα (batch number: CJ44121, manufacturer: Bioworld Technology, Inc., USA) 1 : 500, IKKα (batch number: AA54131, manufacturer: Bioworld Technology, Inc., USA) 1 : 500, and Sheep-Horseradish Peroxidase Anti-Mouse IgG (batch number: 109525, manufacturer: Beijing Zhongshan Biological Technology Co. Ltd., China) 1 : 1000. An automated gel imaging system (manufacturer: Beijing Kechuang Ruixin Biological Technology Co. Ltd., model: K8360, China) was used for the analysis strips.
4
Effects of CoQ10 on Inflammatory Markers
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CoQ10 (98%) was obtained from Nanjing Jingzhu Biotech Ltd. Co. (Nanjing, Jiangsu, China). The DMEM culture medium was bought from Gibco-BRL Company (Gaithersburg, MD, USA). Antibodies specific for IL-6,TNF-α, ICAM-1, VCAM-1, NLRP3, p-AMPK,AMPK,YAP and β-actin were obtained from Proteintech Group (Wuhan, Hubei, China). Antibodies specific for p-YAP and OPA-1 were bought from Abcam (Cambridge, MA, USA).
5
Protein Expression Analysis via Western Blot
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Western blot analysis was performed as previously described.15 The following primary antibodies used were: anti‐VEGFR1, anti‐VEGFR2, anti‐CD31 (Abcam); anti‐VE‐cadherin, anti‐ZO‐1 (zonula occludens‐1), anti‐β‐actin (Sigma‐Aldrich), and anti‐α‐fetoprotein (AFP, R&D systems); Vascular cell adhesion molecule‐1 (VCAM‐1, Cell Signaling Technology); intercellular cell adhesion molecule‐1 (ICAM‐1, Proteintech). Horseradish peroxidase (HRP)‐bound anti‐rabbit IgG (GE Healthcare Biosciences) and the ECL Prime Western Blotting Detection kit (GE Healthcare Biosciences) were also used for observation.
6
Chondroprotective Effects of Signaling Inhibitors
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Reagent sources were as follow: Chick type II collagen (CII) and Freund adjuvant (CFA) were obtained from Chondrex (CII; Chondrex, Remond, WA, USA). The p38 inhibitor SB203580, the JNK inhibitor SP60012 and the ERK inhibitor PD98059 were purchased from Calbiochem (San Diego, CA). Antibodies COX2, GATA4, GAPDH were obtained from Santa Cruz Biotech (Santa Cruz, CA); VEGF (ab46154) was purchased from Abcam; ICAM-1 was purchased from Proteintech (Proteintech, USA). Antibodies phosphor (p)-ERK1/2 (Thr202/Tyr204) (9101), ERK1/2 (9102), p-JNK (Thr183/Tyr185) (9251), JNK (9252), p-p38 (9216), p38 (9212) were purchased from Cell Signaling technology (Beverly, MA).
7
Protein Extraction and Western Blot Analysis
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Protein from cells was extracted with RIPA buffer (Solarbio, Beijing, China). The sample was loaded onto 12% SDS-PAGE gel for electrophoresis and then transferred onto a PVDF membrane. After blocking by 5% skim milk at room temperature for 2 h, the membrane was incubated with primary antibody targeting NPR1 (1:2000, Thermo Fisher Scientific, Waltham, MA, USA), ITGB4 (1:1000, Proteintech, Wuhan, Hubei, China), ICAM-1 (1:1000, Proteintech, Wuhan, Hubei, China), and GAPDH (1:10,000, Proteintech, Wuhan, Hubei, China) at 4 °C overnight. Afterwards, incubation of secondary antibody HRP goat anti-rabbit IgG (1:10,000, Proteintech, Wuhan, Hubei, China) or HRP goat anti-mouse IgG (1:10,000, Proteintech, Wuhan, Hubei, China) was carried out at room temperature for 2 h. The protein band was detected by Tanon 5200 Chemiluminscent and Fluorescent Imaging System (Tanon, Shanghai, China).
8
Protein Expression Analysis in Transfected Cells
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The transfected cells were added to RIPA lysate and protease inhibitor to extract total protein (EpiZyme, Shanghai, China). After lysis on ice for 30 min, the proteins were centrifuged at 12000 rpm for 15 min at 4°C. The loading volume of each sample was measured using a BCA kit (GenStar, Beijing, China). Identical masses of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the proteins were transferred to PVDF membranes (Bio-Rad, UK) and sealed with skim milk powder. PVDF membranes were incubated overnight at 4°C with primary antibodies against VCAM-1 (1:500, Proteintech, USA), ICAM-1 (1:2500, Proteintech, USA), CDH2 (1:1500, Proteintech, USA), and GAPDH (1:10000, Proteintech, USA). Finally, the HRP-labeled goat anti-mouse secondary antibody (1:5000, Proteintech, USA) was incubated with the filter membrane for 1 h. GNG12 protein expression levels were detected using an imager and a chemiluminescent substrate (ECL) kit (Thermo Fisher Scientific, USA).
9
Immunohistochemical Analysis of Ischemic Cortex
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Paraffin sections (2 μm in thickness) were used for all staining. Briefly, following blocking endogenous peroxidase with PBS containing 3% hydrogen peroxide for 5 min, sections were stained with primary antibodies for neutrophils (myeloperoxidase, MPO, Cat#: bs-4943R, 1:200, Bioss, Boston, MA, USA), macrophages (F4/80, Cat#: 28463-1-AP, 1:1000, Proteintech, Wuhan, Hubei, China), intercellular adhesion molecules-1 (ICAM-1, Cat#: 10020-1-AP, 1:1000, Proteintech), vascular adhesion molecule-1 (VCAM-1, Cat#: BM4289, 1:1000, BOSTER, Wuhan, Hubei, China), and P-gp (Cat#: 13978S, 1:1000, Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. Then, sections were incubated with Immunohistochemical staining kit (DAKO, Carpinteria, CA, USA) for 1 h at room temperature. All stained sections were counterstained with Giles’ hematoxylin and imaged on a fluorescent inverted microscope (Ts2R, Nikon). To quantify P-gp expression levels, neutrophil and macrophages, randomly selected fields from the ischemic cortex were analyzed by using ImageJ software.
10
Western Blotting Assay Protocol
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Western blotting assays were carried out as described previously (Du et al., 2017 (link)). Primary antibodies in the experiments were applied as follows: ICAM-1 (diluted at 1:1,000, ProteinTech, Chicago, IL, United States), VCAM-1 (diluted at 1:1,000, ProteinTech), E-selectin (diluted at 1:1,000, R&D Systems, Minneapolis, MN, United States), β-tubulin (diluted at 1:1,000, ProteinTech), NF-κB-p65 (diluted at 1:1,000, CST, Danvers, MA, United States), NF-κB-pp65 (diluted at 1:1,000, CST), IκBα (diluted at 1:1,000, CST), and phospho-IκBα (diluted at 1:1,000, CST). Then the secondary antibodies with horseradish peroxidase coupling (1:10,000 dilution) obtained from ProteinTech were used for 2 h at room temperature. Electrochemiluminescence (ECL) detection reagents were purchased from Millipore (Billerica, MA, United States). Target proteins in the membrane were visualized with a Bio-Rad (Hercules, CA, United States) exposure imaging system.
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