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        Immunohistochemistry Protocol-Cytokine Antibodies

        互联网

        1923
        Introduction
        Immunohistochemistry is used to identify the location
        and distribution of target antigens in cells or
        tissues by staining with a specific antibody. The
        antibody is conjugated to either a fluorescent or
        colorimetric label, and the location of the label
        seen through a microscope approximates the position
        of the target antigen. We present here a
        method for staining of cervical tissue.
        Purpose
        Procedure for indirect staining of cytokines in
        paraformaldehyde fixed cryostat sections with
        monoclonal antibodies.
        Procedure from Walid Al-Saleh Thesis (ULG, Pr.
        Boniver Service of Anatomo-pathology), Liege,
        Belgium. The cytokines IL-2, IL-4, IL-6, IL-10, IL-12,
        IFN- γ and TNF-α proteins in cervical biopsies were
        detected by an original immunohistology technique.
        Materials and Equipment
        • Frozen sections of sample tissues
        • Refrigerator (4ºC)
        • Glass slides
        • Primary antibody
        • Coverslips
        • Secondary antibody
        • 4% Paraformaldehyde, pH 7.4
        • Hematoxylin
        • 1% Goat Serum in TBS/Saponin
        •Ethanol
        • Diaminobenzidine (DAB) substrate solution
        • Microscope
        • Tris-buffered Saline, 0.1% Saponin, pH 7.4
        • Tris-buffered Saline/0.3% H2O2/0.1% Saponin,
        0.02% NaN3
        • Avidin/Biotin/Peroxidase
        (Vectastain, Vector Labs)
        Protocol
        1. Dry frozen tissue sections
        of 8 μm thickness
        at room temperature
        for 2 hours.
        2. Fix sections with 4% paraformaldehyde for 15 minutes at room temperature.
        3. Wash slides 2X, 4 minutes each, with TBS/0.1% saponin.
        4. Block endogenous peroxidase by incubating 30 minutes
        in TBS/0.3% H2O2/0.1% Saponin, 0.02% NaN3.
        5. Wash slides 3X, 3 minutes each, with TBS/saponin.
        6. Block non-specific binding sites with 1/100
        diluted goat serum in TBS/saponin for 20 minutes.
        7. Incubate overnight at 4ºC, with the appropriate antibody.
        8. Wash slides 4 times in TBS/saponin, incubate
        the slides with biotinylated secondary antibody for 30 minutes.
        9. Add avidin-biotin-peroxidase reagents, and
        reveal the resulting peroxidase activity by
        incubating the slides with a 0.5 mg/mL
        HRP substrate solution (DAB + H2O2 prepared in distilled water).
        11. Counterstain for 1 minute with hematoxylin.
        12. Dehydrate slides with sequential ethanol
        washes of 1 minute each starting with
        75%, followed by 80% and finishing with
        a 100% ethanol wash.
        13. Seal slides.
        14. Analyze by optical microscopy.
        Note: It is not necessary to add detergent to the TBS buffer. The
        protocol can be followed s is with saponin omitted from all buffers
        when staining CDs
        <center> <p>  </p> </center>
        上一篇:IHC Protocol for Free Floating Brain Sections   下一篇:IMMUNOHISTOCHEMISTRY on PARAFFIN OR CRYOSECTIONS
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