ArticleThe journal of histochemistry and cytochemistry : official journal of the Histochemistry Society2008
Reduction of high background staining by heating unfixed mouse skeletal muscle tissue sections allows for detection of thermostable antigens with murine monoclonal antibodies.
Article in The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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9 citing papers in PubMed, 18 citations in OpenAlex.
- Verification of Protein Changes Determined by 2D-DIGE Based Proteomics Using Immunofluorescence Microscopy.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Proteomic profiling of liver tissue from theClinical proteomics · 2018Article
- B4GALNT2 (GALGT2) Gene Therapy Reduces Skeletal Muscle Pathology in the FKRP P448L Mouse Model of Limb Girdle Muscular Dystrophy 2I.The American journal of pathology · 2016Article
- A One-Step Immunostaining Method to Visualize Rodent Muscle Fiber Type within a Single Specimen.PloS one · 2016Article
- Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts fromProteomes · 2015Article
- Article
- Merlin isoform 2 in neurofibromatosis type 2-associated polyneuropathy.Nature neuroscience · 2013Article
- Unmasking potential intracellular roles for dysferlin through improved immunolabeling methods.The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society · 2011Article
- Application of WGA lectin staining for visualization of the connective tissue in skeletal muscle, bone, and ligament/tendon studies.Microscopy research and technique · 2011Article
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5 authors at 1 institution in 1 country.
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Abstract
Antigen detection with indirect immunohistochemical methods is hampered by high background staining if the primary antibody is from the same species as the examined tissue. This high background can be eliminated in unfixed cryostat sections of mouse skeletal muscle by boiling sections in PBS, and several proteins including even the low abundant dystrophin protein can then be easily detected with murine monoclonal antibodies. However, not all antigens withstand the boiling procedure. Immunoreactivity of some of these antigens can be restored by subsequent washing in Triton X-100, whereas immunoreactivity of other proteins is not restored by this detergent treatment. When such thermolabile proteins are labeled with polyclonal primary antibodies followed by dichlorotriazinylaminofluorescein-conjugated secondary antibodies and boiled, the fluorescence signal persists, and sections can then be processed with a monoclonal antibody for double immunostaining of a protein unaffected by boiling. This stability of certain fluorochromes on heating can also be exploited for double immunofluorescence labeling of two different thermostable proteins with murine monoclonal antibodies as well as for combination with Y-chromosome fluorescence in situ hybridization. Our method should extend the range of monoclonal antibodies applicable to tissues derived from the same species as the monoclonal antibodies.
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