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Collagen, Type I (COL1) antibody Primary Antibody

COL1 Reactivity: Cow, Deer, Human, Pig, Rabbit, Rat ELISA, IF, IHC, IHC (fro), IHC (p), IHC (pfa) Host: Mouse Monoclonal COL-1
Independent Validation (1)
Catalog No. ABIN957814
$786.50
Plus shipping costs $45.00
50 μL
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  • Target
    Reactivity
    Cow, Deer, Human, Pig, Rabbit, Rat
    Host
    Mouse
    Clonality
    Monoclonal
    Conjugate
    Un-conjugated
    Application
    ELISA, Immunofluorescence (IF), Immunohistochemistry (IHC), Immunohistochemistry (Frozen Sections) (IHC (fro)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunohistochemistry (Paraformaldehyde) (IHC (pfa))
    Brand
    IHC-plus™
    Specificity
    Recognizes the native (helical) form of collagen type I using ELISA and dot-blot. Does not react with the thermally-denatured molecule. The antibody shows no cross-reactivity with collagen types II, III, IV, V, VI, VII, IX, X and XI.
    Purification
    Ascites
    Immunogen
    Full length bovine native protein (purified). Epitope: The epitope recognized by the antibody may be sensitive to routine formalin fixation and paraffin embedding.

    Type of Immunogen: Native protein
    Clone
    COL-1
    Isotype
    IgG1
  • Application Notes
    Approved: ELISA, IF, IHC, IHC-Fr, IHC-P (1:50), IHC-Pfa

    Usage: Suitable for use in ELISA, Western Blot, Immunofluorescence and Immunohistochemistry. In testing of acetone-fixed or unfixed frozen sections, strong staining of connective tissue fibers is seen. Epitope can be sensitive to formalin fixation, will not work with over fixed tissues.
    Comment

    Target Species of Antibody: Bovine

    Restrictions
    For Research Use only
  • Validation #103250 (Immunofluorescence)
    'Independent Validation' Badge
    by
    Musculoskeletal Gene Therapy Research Laboratory, Mayo Clinic
    No.
    #103250
    Date
    11/02/2018
    Antigen
    COL1
    Lot Number
    113240
    Method validated
    Immunofluorescence
    Positive Control

    Rabbit tendon: COL1 is the most abundant collagen in tendons

    Negative Control

    Autofluorescence/endogenous tissue background staining control. Before applying the primary antibody, the rabbit tissues were inspected under the microscope using fluorescence illumination to ensure there was no signal inherent to the tissue itself. Rabbit tissues showed weak endogenous autofluorescence.

    Rabbit cartilage: in normal cartilage, type II collagen represents 90% to 95% of the collagen, whereas COL1 expression is almost negligible.

    Secondary antibody only control.

    Mouse IgG1 kappa isotype control.

    Notes

    Passed. The COL1 antibody ABIN957814 specifically labels the target antigen in IF on FFPE rabbit tendon.

    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN957814
    Secondary Antibody
    biotinylated goat anti-mouse IgG1 antibody (NBP1-69914B, Novus Biologicals)
    Full Protocol
    • Cut formalin-fixed paraffin-embedded (FFPE) rabbit tissues into 5µM sections using a microtome.
    • Deparaffinize and rehydrate by immersing the slides through the following solutions
      • Xylene, wash sections 3x 5min each
      • 100% Ethanol, wash sections 2x 3min each
      • 95% Ethanol, wash sections 2x 1min each
      • 70% Ethanol, wash sections 2x 1min each
      • Deionized Water, wash sections 2x for 5 min each
    • Protease-induced epitope retrieval protocol (0.1% hyaluronidase with 0.1% pronase in phosphate-buffered saline, PBS) using a microwave tissue processor (Milestone) set to 37°C for 30min.
    • Wash the sections by immersing them in distilled water for 5min.
    • Incubate tissue sections with 5% normal goat serum (Abcam, ab7481) in PBS containing 1% Tween 20 (PBST) for 30min at RT.
    • Blot excess serum from the section but do not rinse.
    • Incubate sections with Streptavidin-Biotin blocking solution (SP-2002, Vector Laboratories, Inc) for 15min following the manufacturer's recommendations.
    • Incubate sections with
      • 100µl primary mouse anti-Collagen type I (COL1) antibody (antibodies-online, ABIN957814, lot 113240) diluted 1:50 in PBST ON at 4°C.
      • primary mouse IgG1 kappa isotype control antibody (Abcam, Ab18443) diluted 1:50 in PBST ON at 4°C.
    • Wash sections 3x with PBS-T for 5min each.
    • Incubate sections with the secondary biotinylated goat anti-mouse IgG1 antibody (NBP1-69914B, Novus Biologicals) in a humidified chamber at RT for 1h.
    • Wash sections 3x with PBS-T for 5min each.
    • Tap off excess wash buffer.
    • Incubate sections in fluorescein-streptavidin conjugate (Vector Laboratories Inc., SA-5001) for 5min at RT in a dark slide moat.
    • Wash sections 3x with PBS-T for 5min each.
    • Incubated sections with biotinylated anti-streptavidin antibody (Vector Laboratories Inc., BA-0500) for 30min at RT in a dark slide moat.
    • Incubate the sections in fluorescein-streptavidin conjugate (Vector Laboratories Inc., BA-0500, SA-5001) for 5min at RT in a dark slide moat.
    • Wash sections 3x with PBS-T for 5min each.
    • Apply one drop of Vectashield anti-fade mounting medium with DAPI (Vector Laboratories, Inc, H-1200) and coverslip.
    • Slides can be stored at 4°C in a dark slide box until microscope analysis to prevent loss of fluorescence.
    • Examine slides under a fluorescence automated inverted microscope (Olympus, IX83 microscope).
    Experimental Notes
    • The aim was to demonstrate the performance characteristics of collagen type I (COL1) protein expression using ABIN957814 on FFPE rabbit tissues by using immunofluorescence and that the performance characteristics of the antibody is suitable for the intended analytical use. We decided to use a biotinylated secondary antibody in conjunction with strept-/avidin and a biotinylated reporter in order to enhance the fluorescence signal using the biotinylated goat anti-mouse IgG1 secondary antibody (NBP1-69914B, Novus Biologicals, CO). This technique provided an increased fluorescence intensity.

    • ABIN957814 specifically labeled the target antigen using IF on FFPE rabbit tendon. No signal was detected in negative control tissue (normal rabbit cartilage), isotype control and the secondary antibody only control.

    • We also compared ABIN957814 against another collagen type I antibody from a different company and under similar conditions. ABIN957814 antibody seemed to provide superior immunofluorescence signal.

    • In addition , FFPE sections of normal rabbit condyle were stained using ABIN957814. Normal rabbit cartilage showed no immunofluorescence signal for collagen type 1. The expected COL 1 staining at the subchondral bone was observed.

  • Format
    Liquid
    Concentration
    Lot specific
    Buffer
    15 mM sodium azide.
    Preservative
    Sodium azide
    Precaution of Use
    This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
    Handling Advice
    avoid freeze thaw cycles.
    Storage
    4 °C,-20 °C
    Storage Comment
    Short term 4°C, long term aliquot and store at -20°C, avoid freeze-thaw cycles.
  • Target
    Alternative Name
    Collagen I
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