Protein extracts were obtained from dissected brain structures (cortex, hippocampus, cerebellum) treated with RIPA lysis and extraction buffer (Thermo Fisher Scientific) complemented with SDS powder (5% final; Bio‐Rad, Marnes‐la‐Coquette, France), and the total protein concentration was determined with the BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were denatured at 100°C for 3 minutes, and 25 μg of protein were loaded onto NuPAGE 3–8% Tris‐Acetate Protein gels (Invitrogen), following the manufacturer's instructions. Dystrophin protein was detected by probing the membrane with NCL‐DYS1 primary monoclonal antibody (NCL‐DYS1; Novocastra, Newcastle, UK), and vinculin was detected as the internal control with the hVin‐1 primary antibody (Sigma, St Louis, MO, USA), followed by incubation with a goat anti‐mouse secondary antibody (IRDye 800CW Goat anti‐mouse IgG; Li‐Cor, Germany). Bands were visualized using the Odyssey CLx system (Li‐Cor, Lincoln, NE, USA), and quantification was carried out using the Empiria Studio software (Li‐Cor) based on a standard curve specific of each brain structure and made of a mix of WT and mdx control lysates to obtain defined percentages of dystrophin (0, 5, 10, 15%, or 0, 10, 15, 30% of corresponding WT tissues).
Hvin 1 primary antibody
Manufactured by Merck Group
Sourced in United Kingdom
The HVin-1 primary antibody is a laboratory tool used in research applications. It is designed to detect and bind to a specific target protein or antigen. The core function of this antibody is to enable researchers to identify and study the target of interest within a sample. No further details on intended use or application are provided.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey clx system, by LI COR (3 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (2 mentions)
Nupage 3 8 tris acetate protein gel, by Thermo Fisher Scientific (2 mentions)
Ncl dys1 primary monoclonal antibody, by Leica (2 mentions)
Sds powder 5 final, by Bio-Rad (2 mentions)
Empiria studio, by LI COR (2 mentions)
Empiria studio software, by LI COR (1 mentions)
Ab216343, by Abcam (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Ix51 microscope, by Olympus (1 mentions)
Rhodamine conjugated phalloidin, by Merck Group (1 mentions)
Dapi counterstain, by Vector Laboratories (1 mentions)
Axiophot, by Zeiss (1 mentions)
Fitc conjugated streptavidin, by Vector Laboratories (1 mentions)
5 protocols using hvin 1 primary antibody
1
Dystrophin Quantification in Mouse Brain
2
Western Blot Analysis of CD38 Protein
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Protein extracts were obtained from pooled muscle sections treated with RIPA lysis and extraction buffer (Thermo Fisher Scientific, USA) complemented with SDS powder (5% final) (Bio‐Rad, France), and the total protein concentration was determined with the BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Samples were denatured at 100°C for 3 min, and 7.5 μg of protein was loaded onto NuPAGE 4–12% Bis‐Tris protein gels (Invitrogen) following the manufacturer’s instructions. CD38 protein was detected by probing the membrane with an anti‐CD38 primary polyclonal rabbit antibody (1/1000; ab216343; Abcam, Cambridge, UK), and vinculin was detected as internal control with the hVin‐1 primary antibody (1/20000: Sigma), followed by incubation with a goat anti‐rabbit secondary antibody (1/5000; IRDye 800CW Goat anti‐Rabbit IgG, Li‐COR, Germany). Bands were visualized using the Odyssey CLx System (Li‐COR, Germany). Quantifications were achieved with corresponding WT tissues and normalized to internal control (vinculin) (Empiria Studio, Li‐COR, Germany).
3
Quantitative Dystrophin Protein Analysis
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Protein extracts were obtained from pooled muscle sections treated with radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Thermo Fisher Scientific, USA) complemented with SDS powder (5% final) (Bio-Rad, France), and the total protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA). Samples were denatured at 100°C for 3 min, and 20 μg of protein was loaded onto NuPAGE 3%–8% Tris-acetate protein gels (Invitrogen), following the manufacturer’s instructions. Dystrophin protein was detected by probing the membrane with NCL-DYS1 primary monoclonal antibody (Novocastra, Newcastle, UK), and vinculin was detected as an internal control with the hVin-1 primary antibody (Sigma), followed by incubation with a goat anti-mouse secondary antibody (IRDye 800CW goat anti-mouse IgG, LI-COR Biosciences, Germany). Bands were visualized using the Odyssey CLx system (LI-COR Biosciences, Germany). The quantification was done using a standard curve with 0%, 10%, 20%, 30%, 40%, and 60% of the corresponding WT tissues and normalized to an internal control (vinculin) (Empiria Studio, LI-COR Biosciences, Germany).
4
Evaluating HCMECs Adhesion on Surfaces
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HCMECs were seeded on top of material discs and grown for 24 h under standard cell culture conditions. TCPS plates were used as controls for ideal cell growth. For cell viability monitoring cells were stained using the Live/Dead Cell Staining Kit II (PromoKine, Heidelberg, Germany) according to the manufacturer`s protocol followed by fluorescence microscopy using the IX 51 microscope (Olympus, Hamburg, Germany). Average cell numbers were counted from at least 10 different images per sample type. For visualization of cell adhesion and morphology immunofluorescent staining was conducted. Vinculin was stained using hVIN‐1 primary antibody (Sigma‐Aldrich) and AlexaFluor 488‐coupled anti‐mouse (Molecular Probes, Thermo Fisher Scientific) as secondary antibody. F‐actin was stained using PromoFluor 546‐coupled phalloidin (PromoKine) followed by DAPI staining (PromoKine). Alignment of cell nuclei was quantified by ImageJ, using images of 3 to 5 different areas for each type of surface.
5
Immunofluorescence Staining of Cultured Cells
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After 4 days of culture, cells were fixed (10 mL 37% formaldehyde, 2 g sucrose in 90 mL PBS solution) for 15 minutes. Permeabilising buffer (10.3 g sucrose, 0.292 g NaCl, 0.06 g MgCl2, 0.476 g HEPES, 0.5 mL Triton X, in 100 mL of H2O, at pH 7.2) was then added for 5 mins at 4°C. To block non-specific binding, samples were next incubated in 1% BSA/PBS for 5 mins at 37°C. H-vin 1 primary antibody (1:200, monoclonal antihuman raised in mouse (IgG1) Sigma Aldrich UK, in 1% BSA/PBS) was added for 1 hour along with rhodamine-conjugated phalloidin (1:100, Sigma Aldrich UK). Substrates were then washed three times in 0.5% Tween 20/PBS (5 minutes each). Secondary, biotin-conjugated antibody (1:50 in 1% BSA/PBS, antimouse (IgG) raised in horse, Vector laboratories UK) was added for 1 hour, followed by substrate washing as described above. FITC-conjugated streptavidin was added (1:50 in 1% BSA/PBS, Vector Laboratories UK) for 2 hours before samples were given a final wash. Samples were mounted using mounting medium for fluorescence, with DAPI counterstain (Vector Laboratories), and viewed by fluorescent microscopy (Zeiss Axiophot).
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