After 6 h stroke in mice, ischemic and non-ischemic hemispheres of brains were snap frozen in liquid nitrogen and stored at −80 °C until further use for immunoblotting of fibrin(ogen) and MMP-9 [26 (link)-28 (link)]. Brain tissue (50 mg) was homogenized in lysis buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) containing 1.5 % SDS and 25 mM EACA (ε-aminocaproic acid) (for plasmin inhibition). Protein was estimated by bicinchoninic acid assay using homogenized tissue supernatant after centrifugation. Proteins (50 μg) were electrophoresed on 10% reducing SDS-PAGE gels and transferred to polyvinylidenedifluoride membrane. The membranes were blocked with 1% BSA in PBS containing 0.1% Tween 20. Fibrin(ogen) and MMP-9 were probed with rabbit anti-mouse fibrinogen antibody (MyBiosource) at 1:3000 dilution and goat anti-mouse MMP-9 (1:1000) antibodies respectively, overnight at 4 °C and, detected with anti-rabbit and anti-goat IRDye Li-COR secondary antibodies (LI-COR biosciences, NE). The membranes were scanned on LI-COR Odyssey scanner and pixel density was measured by NIH Image J software. Gelatinase zymography was performed as described [26 (link)].; additional details are available in the Supplemental Data .
Irdye secondary antibody
Manufactured by LI COR
Sourced in United States, Germany, United Kingdom, China
IRDye secondary antibodies are fluorescently labeled antibodies that bind to primary antibodies, enabling visualization and detection of target proteins in various applications such as Western blotting, immunohistochemistry, and flow cytometry. These antibodies are available in a range of fluorescent dye options, providing flexibility in experimental design and detection.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (57 mentions)
Odyssey blocking buffer, by LI COR (43 mentions)
Odyssey clx imaging system, by LI COR (30 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (25 mentions)
Odyssey imaging system, by LI COR (21 mentions)
Image studio software, by LI COR (18 mentions)
Odyssey imager, by LI COR (17 mentions)
Bca protein assay, by Thermo Fisher Scientific (16 mentions)
Odyssey clx infrared imaging system, by LI COR (16 mentions)
Protease inhibitor cocktail, by Merck Group (14 mentions)
Odyssey clx imager, by LI COR (12 mentions)
Nitrocellulose membrane, by Bio-Rad (12 mentions)
Odyssey clx, by LI COR (12 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
β actin, by Merck Group (11 mentions)
Trans blot turbo transfer system, by Bio-Rad (10 mentions)
Odyssey fc imaging system, by LI COR (9 mentions)
Blocking buffer, by LI COR (9 mentions)
Nitrocellulose membrane, by GE Healthcare (9 mentions)
Pvdf membrane, by Merck Group (8 mentions)
362 protocols using irdye secondary antibody
1
Quantifying Fibrin(ogen) and MMP-9 in Ischemic Stroke
2
Protein Analysis by Western Blot and Immunostaining
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Western blot was performed as described previously48 (link). Briefly, treated cells or Exos were lysed and suspended in Tris-glycine sample buffer with proteinase inhibitor EDTA-free cocktail. Equal amounts of lysates were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. Blots were blocked followed by incubation with primary antibodies (Supplementary Table S2 ) overnight at 4 °C. IRDye LiCor secondary antibodies (Li-Cor Biosciences) were used for protein detection with LiCor Odyssey CLX imaging system (Li-Cor Biosciences). Quantification of protein bands was done with Image Studio. Immunostaining was performed as described previously57 (link). Isotype control (IgG2a kappa) was used as a negative control for K15 antibody.
3
Western Blot Analysis of Proteins
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SDS-PAGE was carried out on 7.5% acrylamide gels or 4–20% gradient gels under reducing conditions. Proteins were transferred to Immobilon-FL transfer membranes (EMD Milipore, cat. no. IPFL00010) and blocked using 5% nonfat dry milk made in PBST. Primary antibodies were diluted in blocking buffer and incubated overnight at 4 °C. LI-COR IR dye secondary antibodies (LI-COR Biosciences, Lincoln, NE), goat anti-mouse IR dye 800CW (Catalog no. 926-32210), or goat anti-rabbit IR dye 680RD (Catalog no. 926-68071) were used at 1:10,000 dilutions in PBS to detect primary antibodies. Uncropped images of all blots can be found in Supplementary Fig. 8 .
4
Western Blot Analysis of Synaptic Markers
5
Protein Expression Analysis in Mouse As4.1 Cells
6
Quantifying Brain Protein Levels
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Protein concentrations in brain extracts were measured using the BCA method (Pierce), and equal amounts of protein from the homogenized lysates were loaded onto SDS-PAGE gels and transferred to PVDF membranes. After the membranes were blocked, proteins were detected with one of the following primary antibodies: anti-GFAP (1:500, Millipore), anti-PSD-95 (1:200, Cell Signaling Technology), or anti-synaptophysin (1:200, Millipore). The membranes were probed with LI-COR IRDye secondary antibodies, and proteins were detected using the Odyssey infrared imaging system (LI-COR).
7
H2A Ubiquitylation Assay Protocol
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H2A ubiquitylation assays were carried out as previously (Rose et al., 2016 ). Briefly, UBE1 (Boston Biochem), UbcH5c (Enzo), methylated ubiquitin (Boston Biochem) and ATP (Life technologies) were pre-incubated for 20 min at 37°C prior to addition of reconstituted PRC1 and nucleosomes. Reactions were allowed to proceed for 1 h at 37°C then quenched with 30 mM EDTA and subject to SDS-PAGE for western blot analysis. Western blots were probed with antibodies which recognize Histone H2A in both ubiquitylated and unmodified form (Millipore 07-146) and Histone H3 (CST, 96C10), followed by incubation with LiCOR IRDye secondary antibodies (800CW goat anti-rabbit and 680RD goat anti-mouse). Western blots were imaged using the LiCOR Odyssey Fc imaging system and band intensities were quantified using ImageStudio. H2A band intensities were normalized to H3 and the fraction of ubiquitylated H2A relative to total H2A was quantified. Data were visualized and dose-response curves fitted using GraphPad Prism 7.
8
Western Blot Analysis of Muscle Proteins
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Briefly, muscles were lysed with bead homogenizer (Precelly 24 lysis & homogenization, Bertin technologies) in ice-cold RIPA buffer: 25 mM Tris pH 7.6, 150 mM NaCl, 1 mM Na3VO4, 10 mM NaPyroPO4, 10 mM β-glycerophosphate, 10 mM NaF, 1 mM PMSF, 1X protease inhibitor cocktail (Santa Cruz), 1% NP40, 1% sodium deoxycholate and 0.1% SDS. After quantification by BCA protein assay (Thermo Scientific) equal amounts of muscle homogenates were resolved by SDS-PAGE, transferred to PVDF membrane (Millipore, Billercia, United States) and probed using primary antibodies from the vendors and at the dilutions shown in Supplementary Table 1 with LI-COR IRDye secondary antibodies (LI-COR Inc, Lincoln, United States). Immunoreactive bands were visualized and quantified by densitometry using the Odyssey Infrared Imaging System and software (LI-COR). To control for variations among different western blots and allow for multiple repeats, the absorbance intensity for each band on the western blot was normalized to the absorbance intensity of corresponding band from the control mice from the same western blot, averaged with values obtained from other western blots and plotted as % control. Some blots had multiple controls and the absorbance of the specific band was normalized to the average56 (link). Full length western blots are show in Supplementary Fig. 9 .
9
Western Blot Protein Quantification Protocol
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Cell pellets were collected and lysed (mammalian PER, Thermo scientific + 1x protease inhibitor cocktail, Thermo Scientific), and samples were prepared after protein quantification. We loaded about 15 µg protein per lane of a polyacrylamide gel (NuPAGE™ Novex™ 4–12% Bis-Tris Protein Gels). Once the gels were resolved, they were transferred onto nitrocellulose membrane and subsequently blocked in 5% milk solution for a minimum of 2 h. This was followed by a one-step i-Bind process which treated the membrane with primary antibody, washing and secondary antibody steps (Life technologies). We employed LiCor® IRDye secondary antibodies (680 and 800 wavelength infrared dyes) and detection of bands was carried out in a LiCor ODyssey CLx imager (LiCor). Antibodies and dilutions employed can be found in Supplementary Table 3 . Full blots are provided in Supplementary Figs. 4 and 5 .
10
Immunoblot Analysis of Protein Regulation
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