Transverse frozen sections were cut from the mid-belly of the TA or diaphragm muscle strips at a thickness of 8 μm, mounted on slides and stored at −80 °C until analysis. Immunohistochemistry for ADAMTS1 (Origene, TA317919, Rockville, MD, USA), ADAMTS5, ADAMTS15 (Abcam, ab45047, Cambridge, MA, USA), V0/V1 versican (anti-GAGβ; Merck Millipore, AB1033, Bayswater, VIC, Australia) or versikine (anti-DPEAAE neo-epitope; Thermo Fisher Scientific, PA1-1748A, Scoresby, VIC, Australia) were performed as previously described [9 (link),32 (link)]. An anti-desmin rabbit polyclonal antibody (Abcam, ab15200, Cambridge, MA, USA) together with an anti-rabbit Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific, R37116,) were used to detect myoblasts and newly regenerated myofibres [93 (link),94 (link)]. Representative wild type and mdx TA and diaphragm muscle cross-sections were H and E stained for muscle architecture, and wheat germ agglutinin to assess fibrosis [95 (link)]. For analysis of V0/V1 versican and versikine immunoreactivity, four non-overlapping representative digital images were captured with a confocal microscope of each muscle section at 600× magnification (Olympus Fluoview FV10i) and analysed for area of immunoreactivity using Image-Pro Plus software (Version 7, Media Cybernetics, Silver Spring, MD, USA).
Ab1033
Manufactured by Merck Group
Sourced in United States
The AB1033 is a laboratory instrument designed for analyzing molecular samples. It utilizes advanced spectroscopic techniques to detect and measure the properties of various biomolecules. The core function of the AB1033 is to provide precise and reliable data for scientific research and clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse versican, by Merck Group (2 mentions)
Biotin sp conjugated goat anti rabbit igg secondary antibody, by Jackson ImmunoResearch (2 mentions)
Af488 conjugated donkey anti sheep igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
Spot rt3 microscope, by Olympus (2 mentions)
Anti human fibronectin, by Merck Group (2 mentions)
Rabbit igg isotype control, by Santa Cruz Biotechnology (2 mentions)
Af3715, by Abcam (2 mentions)
Cm3050s cryostat, by Leica camera (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab15200, by Abcam (1 mentions)
Ab45047, by Abcam (1 mentions)
Anti dpeaae neo epitope, by Thermo Fisher Scientific (1 mentions)
Fluoview fv10i, by Olympus (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
H3506, by Merck Group (1 mentions)
Nova red, by Agilent Technologies (1 mentions)
Ab1032, by Merck Group (1 mentions)
C3667, by Merck Group (1 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
5 protocols using ab1033
1
Immunohistochemical Analysis of ADAMTS Proteases and Versican in Skeletal Muscle
2
Immunohistochemical Analysis of Tumor Tissues
3
Immunohistochemical Analysis of Tumor Tissues
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OCT-embedded, frozen tumor tissues were cryosectioned in a Leica CM3050 S Cryostat at 10μm thickness for all immunohistochemical (IHC) and immunofluorescence (IF) studies. Slides were incubated overnight in primary antibody at 4°C. Secondary/tertiary antibodies were added and sections were developed and counterstained according to the appropriate protocol. For IHC, the following antibodies were used: anti-human fibronectin (Sigma #F3648), anti-mouse versican (EMD Millipore #AB1033, Burlington, MA, USA), biotinylated hyaluronic acid binding protein, HABP (EMD Millipore #385911), Rabbit IgG isotype control (Santa Cruz Biotechnology #sc-2027, Dallas, TX, USA), and biotin-SP-conjugated goat anti-rabbit IgG secondary antibody (Jackson Immunoresearch #AB2337959, West Grove, PA, USA). The following antibodies were used for IF: anti-human FAP antibody (R&D Systems #AF3715, Minneapolis, MN, USA or Abcam #53066, Cambridge, MA, USA) or sheep IgG isotype control (R&D Systems #5001A), and AF488-conjugated donkey anti-sheep IgG secondary antibody (ThermoFisher #A11015, Waltham, MA, USA). All imaging was completed with the Olympus Spot-RT3 microscope at 20X magnification. For quantification purposes, 3-5 different tumor samples (n≥3) from each experimental group were used. The mean area of positive staining and mean fluorescence intensity were determined using Fiji ImageJ Software.
4
Quantitative Analysis of Extracellular Matrix Proteins
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Sections from lateral tendon proximal to the lesion were prepared from 2 randomly selected control and transected tendons (two regions per tendon: one adjacent and one most distant to the lesion), and pretreated with either hyaluronidase (Sigma #H3506, 1000U/mL for one hour at 37°C; slides for aggrecan) or chondroitinase ABC (Sigma #C3667, 0.2U/mL for two hours at 37°C; slides for versican, biglycan and fibromodulin). Slides were washed and then incubated (4°C overnight) with affinity-purified rabbit polyclonal antibodies to the G1 region of aggrecan (provided by Dr John Mort, McGill University, Canada[28 (link)], 1:1000), the GAG-alpha and GAG-beta regions of versican (Millipore AB1032 and AB1033, respectively, both at 5μg/mL), and the human C-terminal peptide sequences of biglycan and fibromodulin (EF2 and EF3 respectively, generated by Dr Emily Fuller, University of Sydney, both at 1μg/mL). Antibody binding was detected with rabbit Envision (Dako, NovaRed) for 30 minutes at room temperature then counterstained with Mayer’s heamatoxylin.
5
Analyzing Proteome Changes in FLC and NML Tissues
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FLC and NML tissues were lysed in RIPA buffer containing Halt protease and phosphatase inhibitors (Thermo Fisher Scientific) at 4°C. Lysates were incubated for 30 minutes and centrifuged at 14,000 × g for 10 minutes at 4°C. Total protein in the supernatant was quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were denatured in NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) containing 5% β-Mercaptoethanol for 10 minutes at 70°C and loaded to a 12% SDS-polyacrylamide gel. After electrophoresis, samples were transferred to polyvinylidene difluoride membrane and blocked in TBS containing 0.5% TWEEN20 (TBST) and 3% BSA for 1 hour at room temperature. Membranes were probed for anti-VCAN GAGβ (1:1,000 dilution, rabbit source, Millipore AB1033) or anti-vinculin (1:10,000 dilution, mouse source VLN01, Thermo Fisher Scientific MA5-11690) overnight at 4°C and then incubated with goat anti-rabbit horseradish peroxidase–linked IgG (1:10,000, Cell Signaling Technology). Membranes were visualized using a ChemiDoc MP (Bio-Rad).
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