Lung sections were prepared as previously described, then stained with mouse anti-MUC5AC antibody (#ab3649, Abcam), rabbit anti-α-SMA (#ab32575, Abcam), TLR4 (#ab13556, Abcam), rabbit anti-HMGB1 antibody (#ab79823, Abcam), rabbit anti-NLRP3 antibody (#214185, Abcam), rabbit anti-NLRC4 antibody (#A13117, ABclonal) and rabbit anti-Bcl-2 antibody (#A0208, ABclonal). After washing with PBS, slices were incubated with anti-rabbit secondary antibody for 30 minutes at room temperature. Signals were visualized with a DAB peroxidase substrate kit., and the localization of targeted molecules in the lung was assessed under a light microscope. Lung tissue samples were ground to fine powder under liquid nitrogen and lysates were subjected to SDS-PAGE and western blotting for the detection of the following antigens: TLR4 (#ab13556, Abcam), HMGB1 (#ab79823, Abcam), NLRP3 (#214185, Abcam), NLRC4 (#A13117, ABclonal), Bcl-2 (#A0208, ABclonal), procaspase-3 (#A0214, ABclonal), procaspase-8 (#108333, Abcam), cleaved caspase-3 (#9664s, Cell Signaling Technology) and cleaved caspase-8 (#9429, Cell Signaling Technology). After incubation with an IRDye® 680WC-conjugated secondary antibody (LICOR Biosciences), immunoreactive bands were exposed to an Odyssey® CLx Imager for image capture. Data analysis was performed with ImageJ Software.
Irdye 680wc conjugated secondary antibody
Manufactured by LI COR
The IRDye® 680WC-conjugated secondary antibody is a fluorescently labeled secondary antibody. It is designed for use in immunoassays and other applications that require detection of primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey clx imager, by LI COR (4 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Ab3649, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Ab79823, by Abcam (1 mentions)
Ab13556, by Abcam (1 mentions)
4 protocols using irdye 680wc conjugated secondary antibody
1
Molecular Profiling of Lung Inflammation
2
Western Blot Analysis of RAGE, HDAC1, AKT Signaling
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In order to evaluate the expression of RAGE, HDAC1, AKT, p-AKT, E-cadherin, and β-catenin in vivo and in vitro, whole lung tissue and cell protein extracts were mixed with 5× SDS loading buffer. The samples were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). Then the membranes were probed with anti-RAGE, anti-HDAC1, anti-AKT, anti-p-AKT, anti-E-cadherin, and anti-β-catenin antibodies using the recommended dilutions. The immunoreactive bands were imaged using an Odyssey® CLx Imager after incubation with an IRDye® 680WC-conjugated secondary antibody (LI-COR Biosciences). Odyssey software was used for data analysis, and Image J software was used for quantitative image analysis.
3
Protein Expression Analysis of Key Markers
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To evaluate the protein expression of Hsp90α, Hsp90β, E-cadherin, β-catenin, MLC, and p-MLC, Total cell lysates were subjected to 10% or 12% SDS-PAGE, transferred to PVDF membrane (Bedford, MA), and then probed with the following antibodies: Hsp90α, Hsp90β, MLC, p-MLC, E-cadherin and β-catenin. After incubation with an IRDye®680WC-conjugated secondary antibody (LI-COR Biosciences), immunoreactive bands were exposed to Odyssey® CLx Imager for image capture. Or after incubation with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit or anti-mouse IgG antibody, the immunoreactive bands were detected using the enhanced chemiluminescence from Millipore Company (Bedford, MA). Quantitative image analysis was performed with Image J software.
4
Protein Expression Analysis in Lung Tissue
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In order to evaluate the expression of RAGE, HDAC1, AKT, and p-AKT in vivo and in vitro, whole lung tissue and cell protein extracts were mixed with 5× SDS loading buffer and separated by 10% SDS-polyacrylamide gel electrophoresis. The samples were then transferred to PVDF membranes (Millipore), and the membranes were probed with anti-RAGE, anti-HDAC1, anti-AKT, and anti-p-AKT antibodies using the recommended dilutions. After incubation with an IRDye® 680WC-conjugated secondary antibody (LI-COR Biosciences), immunoreactive bands were imaged using an Odyssey® CLx Imager. Odyssey software was used for data analysis, and Image J software was used for quantitative image analysis.
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