Nicotine (cat no. N5768), α-bungarotoxin (α-BTX; a competitive irreversible antagonist of α7nAChR; cat no. T0195) and puromycin (cat no. P9620) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The MEK1/2 inhibitor U0126 (cat no. S1901) was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against ERK (cat no. 4695), phospho-p42/44 ERK (cat no. 4370), vimentin (cat no. 5741), β-actin (cat no. 4970), GAPDH (cat no. 5174) and anti-rabbit IgG, an HRP-linked antibody (cat no. 7074) used for western blotting or immunofluorescence, were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The anti-α7nAChR antibody was purchased from Abcam (Cambridge, MA, USA; cat no. ab24644).
Anti α7 nachr antibody
Manufactured by Abcam
Sourced in United States
The Anti-α7-nAChR antibody is a laboratory research tool used to detect and study the α7 subunit of the nicotinic acetylcholine receptor (nAChR) in various samples. This antibody specifically recognizes the α7 subunit of the nAChR, which is involved in a variety of physiological processes.
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Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
α bungarotoxin α btx, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Puromycin, by Merck Group (1 mentions)
Nicotine, by Merck Group (1 mentions)
U0126, by Beyotime (1 mentions)
Biotinylated anti rabbit igg, by Abcam (1 mentions)
Anti pcna antibody, by Cell Signaling Technology (1 mentions)
Anti fibronectin antibody, by Abcam (1 mentions)
Anti e cadherin antibody, by BD (1 mentions)
Anti zo 1 antibody, by BD (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Mecamylamine hydrochloride mec, by Merck Group (1 mentions)
α bungarotoxin α bgtx, by Bio-Techne (1 mentions)
Bebm media, by Lonza (1 mentions)
5 protocols using anti α7 nachr antibody
1
Investigating α7nAChR-mediated Signaling Pathways
2
Quantifying Hippocampal α7nAChR Expression
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Paraffin sections were used for immunohistochemistry. Serial coronal sections of brain were gained and hippocampal sections were gathered in sequence. Every 15th staining section was chosen for quantitative analysis (five sections per rat). After being dewaxed in xylene, hippocampal sections were hydrated in a graded series of alcohols. Then the sections placed into 0.01 M citrate buffer (pH6.0) were heated for 8 min in microwave oven to complete antigen retrieval, subsequently were blocked for endogenous peroxidases. The treated hippocampal sections were sealed using 3% BSA for 30 min at room temperature, and were then incubated with an anti-α7nAChR antibody (0.5 mg/ml, Abcam, Cat. ab110851, United States ) overnight at 4°C. After washing three times, the sections were incubated with a biotinylated anti-rabbit IgG (1:1,000, Abcam, Cat.150,077, United States ) for 1 h at 37°C and were then developed via DAB reagent. Subsequently, the sections were counterstained with hematoxylin, dehydrated, and mounted on gelatin-coated slides. Images of CA1 and CA3 regions were obtained with 90× magnification. Three fields in one section were chosen for calculating the numbers of positive neurons. Positively-labeled neurons were quantified using ImageJ software.
3
Protein Expression Analysis in Cell Lysates
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Cell lysate combinations of RIPA with PIC2 and PPI were used to form the lysis buffer, and a reaction time of 30 min in an ice bottle was allocated. The protein concentration in each cell lysate was then measured using a commercial BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). In the Western blot analysis, an SDS-polyacrylamide gel electrophoresis system was used. An anti-GAPDH antibody (Cell Signaling, Danvers, MA, USA), anti-fibronectin antibody (Abcam, Cambridge, England, UK), anti-vimentin antibody (cell signaling), anti-E-cadherin antibody (BD Biosciences, Franklin Lakes, NJ, USA), anti-ZO-1 antibody (BD Biosciences), anti-PCNA antibody (Cell Signaling), anti-protein kinase B (AKT) antibody (Cell Signaling), anti-phospho-AKT (P-AKT) antibody (Cell Signaling), anti-α7-nAChR antibody (Abcam), and anti-SNCG antibody (Cell Signaling) were used for probing.
4
Immunohistochemical Staining of α7-nAChR
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An anti-α7-nAChR antibody, which was a rabbit polyclonal antibody specific for human α7 nAChR, was purchased from Abcam Biotechnology (ab10096, USA). The procedure for IHC has been described previously.[24 (link)] Briefly, all sections firstly underwent deparaffinization and rehydration, and were then heated in a 1 mmol/L ethylenediaminetetraacetic acid (EDTA) buffer (water bath, 96–98°C) for 15 minutes in order to retrieve the antigens. A 3% hydrogen peroxide was used to quench the endogenous peroxidase activity, and nonspecific binding was blocked by 10% normal goat serum. We used the primary anti-α7-nAChR antibody at a dilution of 1:250. The primary antibody was replaced by normal serum or phosphate-buffered saline (PBS) as a negative control.
5
Squamous Esophageal Cell Culture and Characterization
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Human Het-1A squamous esophageal cells were obtained from American Type Culture Collection (ATCC, CRL-2692). Cells were cultured at 37°C and 5% CO2 in BEBM media (Lonza/Clonetics Corporation) according to ATCC recommendations. Culture flasks and plates were precoated with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin dissolved in culture medium. Cells were seeded in 96-well plates (1×104 cells per well), and 24 h later the analyzed compounds were added to cells. Next, cells were incubated for 48 h, examined under microscope, and characterized using WST-1 assay and fluorescent Hoechst/propidium iodide assay as described elsewhere [19 (link),25 (link)]. Hoechst/propidium iodide assay allows evaluation of cell viability by staining all cell nuclei with Hoechst 33342 and dead cell nuclei with propidium iodide. For inhibition of nAChRs and muscarinic acetylcholine receptors (mAChRs), 1 h preincubation of keratinocytes with 1 μM α-bungarotoxin (α-Bgtx, Tocris), 10 μM mecamylamine hydrochloride (Mec, Sigma), 1 μM atropine (Sigma), or 1 μg per 50 μl anti-α7-nAChR antibody (#ab10096, Abcam, Cambridge, UK) was performed before rSLURP-1 application. Antibodies at the same concentration were additionally added to the cells after 24 and 40 hours of incubation with rSLURP-1.
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