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  1. Article ; Online: Preparation of Yeast Cells for Staining.

    Rodig, Scott J

    Cold Spring Harbor protocols

    2022  Volume 2022, Issue 6, Page(s) Pdb.prot099648

    Abstract: Staining yeast cells for the presence and location of antigens is particularly challenging. They are small, making the resolution of any antigen difficult; they have a thick cell wall that antibodies cannot penetrate and that is difficult to remove; and ... ...

    Abstract Staining yeast cells for the presence and location of antigens is particularly challenging. They are small, making the resolution of any antigen difficult; they have a thick cell wall that antibodies cannot penetrate and that is difficult to remove; and they grow in suspension, making handling difficult. In addition, background problems can be especially severe, particularly with polyclonal antibodies, because many antisera contain antibodies to yeast cell wall components. In this protocol, yeast cells are treated with paraformaldehyde, the cell wall is removed by enzymic digestion, and the spheroplasts are attached to poly-l-lysine-coated slides. After cell lysis, the cells are ready to be stained as per normal. Except in unusual circumstances, the detection reagent should be fluorochrome-labeled.
    MeSH term(s) Antibodies ; Antigens ; Saccharomyces cerevisiae ; Spheroplasts ; Staining and Labeling
    Chemical Substances Antibodies ; Antigens
    Language English
    Publishing date 2022-06-24
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot099648
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Cell Staining.

    Rodig, Scott J

    Cold Spring Harbor protocols

    2022  Volume 2022, Issue 6, Page(s) Pdb.top099606

    Abstract: When labeled antibodies are used to detect antigens in cells or tissues, several characteristics of an antigen can be readily determined. Most importantly, cell staining will show both the presence and subcellular localization of an antigen. Double- ... ...

    Abstract When labeled antibodies are used to detect antigens in cells or tissues, several characteristics of an antigen can be readily determined. Most importantly, cell staining will show both the presence and subcellular localization of an antigen. Double-labeling techniques permit the simultaneous detection of two antigens, allowing comparisons of the relative distribution of different antigens. Many cell-staining methods can also be used in conjunction with conventional histological stains and autoradiographic methods to compare the localization of the antigen with other markers. Cell staining can also be used in pathology studies for determining such variables as the type of infectious organism, the progenitor of a neoplastic cell, or the presence of an inflammatory response. With certain modifications, the procedure can be used to purify cells on the basis of the antigenic composition of their cell surface. Cell staining is a versatile technique and, if the antigen is highly localized, can detect as few as a thousand antigen molecules in a cell or tissue. In some circumstances, cell staining may also be used to determine the approximate concentration of an antigen. Improvements in antibody labeling methods, microscopes, cameras, and image analyzers are rapidly extending the sensitivity of cell-staining procedures and are making these techniques more quantitative. Even without these improvements, cell staining can yield important qualitative and semiquantitative data. This introduction describes protocols for cell staining techniques and includes a discussion of major constraints, antibody selection, and troubleshooting.
    MeSH term(s) Antibodies ; Antigens ; Staining and Labeling
    Chemical Substances Antibodies ; Antigens
    Language English
    Publishing date 2022-06-24
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.top099606
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Fixing and Binding Antibodies to Suspension Cells in Preparation for Staining.

    Rodig, Scott J

    Cold Spring Harbor protocols

    2021  Volume 2021, Issue 6

    Abstract: Staining of suspension cells after fixation, described here, is normally used only to detect cell-surface antigens. Once cells are fixed and permeabilized, the antibodies are added. The antibodies can be labeled directly or they can be detected by using ... ...

    Abstract Staining of suspension cells after fixation, described here, is normally used only to detect cell-surface antigens. Once cells are fixed and permeabilized, the antibodies are added. The antibodies can be labeled directly or they can be detected by using a labeled secondary reagent that will bind specifically to the primary antibody. Detection reagents for cell staining can be labeled with fluorochromes, enzymes, gold, or iodine. Because the cells are in suspension, the washing steps are tedious, and care should be taken not to centrifuge for long durations or at high speeds.
    MeSH term(s) Animals ; Antibodies/immunology ; Antibodies/metabolism ; Antibody Specificity/immunology ; Antigen-Antibody Reactions/immunology ; Antigens, Surface/immunology ; Antigens, Surface/metabolism ; Cells, Cultured ; Humans ; Protein Binding ; Staining and Labeling/methods ; Tissue Fixation/methods
    Chemical Substances Antibodies ; Antigens, Surface
    Language English
    Publishing date 2021-06-01
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot099697
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Detecting Fluorochrome-Labeled Cells.

    Rodig, Scott J

    Cold Spring Harbor protocols

    2021  Volume 2021, Issue 6

    Abstract: This protocol describes cell staining using fluorochrome-labeled antibodies. The resolution of subcellular structures using fluorochrome-labeled antibodies exceeds that of the transmitted light microscope because of the visualization of an expanding cone ...

    Abstract This protocol describes cell staining using fluorochrome-labeled antibodies. The resolution of subcellular structures using fluorochrome-labeled antibodies exceeds that of the transmitted light microscope because of the visualization of an expanding cone of emitted light from the excited fluorochrome in the specimen. The most commonly used fluorochromes are fluorescein and rhodamine. In recent years, a number of advances in the development of fluorochromes have resulted in brighter and longer-lasting dyes with narrow emission spectra. These are available from a number of commercial suppliers. They can be conjugated to anti-immunoglobulin antibodies, Protein A, Protein G, avidin, or streptavidin. These conjugates are available from many commercial sources. Filter sets are commonly available that will permit independent observation of these two fluorochromes in the same sample. The fluorochrome Texas Red is also used for immunofluorescence, and can be detected using the same filter sets as rhodamine.
    MeSH term(s) Animals ; Antibodies/chemistry ; Cells, Cultured ; Fluorescein/chemistry ; Fluorescent Dyes/chemistry ; Humans ; Microscopy, Fluorescence/methods ; Rhodamines/chemistry ; Staining and Labeling/methods ; Xanthenes/chemistry
    Chemical Substances Antibodies ; Fluorescent Dyes ; Rhodamines ; Xanthenes ; Texas red (82354-19-6) ; Fluorescein (TPY09G7XIR)
    Language English
    Publishing date 2021-06-01
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot099747
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Preparing Frozen Tissue Sections for Staining.

    Rodig, Scott J

    Cold Spring Harbor protocols

    2021  Volume 2021, Issue 3

    Abstract: Cell-staining studies on mammalian tissue are often performed on frozen sections. This is the gentlest method for the preparation of samples and gives good preservation of cell structure and antigens. Its principal disadvantages are that the specimens ... ...

    Abstract Cell-staining studies on mammalian tissue are often performed on frozen sections. This is the gentlest method for the preparation of samples and gives good preservation of cell structure and antigens. Its principal disadvantages are that the specimens must be stored frozen and a special microtome, known as a cryostat, is required. In addition, many clinical specimens are not available in this form, and most classical histological descriptions of tissue structure and pathology are based on the use of formalin-fixed, paraffin-embedded (FFPE) material.
    MeSH term(s) Animals ; Antibodies/metabolism ; Frozen Sections/methods ; Staining and Labeling/methods
    Chemical Substances Antibodies
    Language English
    Publishing date 2021-03-01
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot099655
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Preparing Paraffin Tissue Sections for Staining.

    Rodig, Scott J

    Cold Spring Harbor protocols

    2021  Volume 2021, Issue 3

    Abstract: Most histological studies are performed on formalin-fixed, paraffin-embedded (FFPE) tissue samples. Therefore, there is an extensive atlas of most tissues and organs prepared from these sources, and comparing the location of antigens to these data is ... ...

    Abstract Most histological studies are performed on formalin-fixed, paraffin-embedded (FFPE) tissue samples. Therefore, there is an extensive atlas of most tissues and organs prepared from these sources, and comparing the location of antigens to these data is immediately informative. Fixation and embedding procedures for preparation of paraffin tissue sections are described here. Because of the harsh fixation, embedding, and preparation conditions used in this procedure, many antigens are not well preserved. Thus, cell staining of paraffin-embedded tissue sections usually requires sensitive detection methods and may require amplification using multiple-layer techniques. The protein cross-linking associated with these fixation conditions can mask epitopes. To uncover them and thus improve antibody-antigen binding, the epitopes can be unmasked by reversing the protein cross-linking. One thermal method, heat-induced epitope retrieval, is presented here.
    MeSH term(s) Antigens/metabolism ; Epitopes/metabolism ; Hot Temperature ; Paraffin Embedding/methods ; Staining and Labeling ; Tissue Fixation/methods
    Chemical Substances Antigens ; Epitopes
    Language English
    Publishing date 2021-03-01
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot099663
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: Embedding Cultured Cells in Matrigel for Staining.

    Rodig, Scott J

    Cold Spring Harbor protocols

    2021  Volume 2021, Issue 3

    Abstract: Adherent cells normally are prepared for cell staining by growing on a suitable support. Suspension cells can be fixed directly or can be attached to a solid support by centrifugation, chemical cross-linking, or simple dehydration. An alternative ... ...

    Abstract Adherent cells normally are prepared for cell staining by growing on a suitable support. Suspension cells can be fixed directly or can be attached to a solid support by centrifugation, chemical cross-linking, or simple dehydration. An alternative approach for cells grown in culture is to prepare a fixed cell pellet that is embedded in a scaffold, such as Matrigel, and then processed, embedded in paraffin, and stained in a manner identical to formalin-fixed tissue specimens.
    MeSH term(s) Cell Culture Techniques/methods ; Cells, Cultured ; Collagen/chemistry ; Drug Combinations ; Laminin/chemistry ; Paraffin Embedding/methods ; Proteoglycans/chemistry ; Staining and Labeling
    Chemical Substances Drug Combinations ; Laminin ; Proteoglycans ; matrigel (119978-18-6) ; Collagen (9007-34-5)
    Language English
    Publishing date 2021-03-01
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot099671
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: Detecting β-Galactosidase-Labeled Cells.

    Rodig, Scott J

    Cold Spring Harbor protocols

    2020  Volume 2020, Issue 5, Page(s) 99739

    Abstract: β-Galactosidase has been used extensively both as a label in enzyme immunoassays and for immunocytochemistry. One good substrate is 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal), which gives an intense blue product. The product is stable and ... ...

    Abstract β-Galactosidase has been used extensively both as a label in enzyme immunoassays and for immunocytochemistry. One good substrate is 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal), which gives an intense blue product. The product is stable and insoluble in alcohol as well as H
    MeSH term(s) Galactosides/metabolism ; Immunoenzyme Techniques/instrumentation ; Immunoenzyme Techniques/methods ; Immunohistochemistry/instrumentation ; Immunohistochemistry/methods ; Indoles/metabolism ; Substrate Specificity ; beta-Galactosidase/metabolism
    Chemical Substances Galactosides ; Indoles ; beta-Galactosidase (EC 3.2.1.23) ; 5-bromo-4-chloro-3-indolyl beta-galactoside (V595OG374W)
    Language English
    Publishing date 2020-05-01
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot099739
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article ; Online: Attaching Suspension Cells to Slides for Staining.

    Rodig, Scott J

    Cold Spring Harbor protocols

    2020  Volume 2020, Issue 12

    Abstract: Suspension cells can be prepared for staining by several different methods. A simple method for detecting intracellular antigens in cells that grow in suspension is to attach the cells to a solid substrate before fixation. This can be achieved by the use ...

    Abstract Suspension cells can be prepared for staining by several different methods. A simple method for detecting intracellular antigens in cells that grow in suspension is to attach the cells to a solid substrate before fixation. This can be achieved by the use of a cytocentrifuge. For surface staining, suspension cells can be attached to slides by cross-linking with poly-l-lysine. Lysine can be polymerized to any desired length, and poly-l-lysine will bind to most solid supports through its charged side chains. The positively charged polymer will provide a site for binding of cells (which carry an overall negative charge). Although this cross-link is not covalent, it is sufficiently strong for most cell-staining techniques.
    MeSH term(s) Animals ; Cell Adhesion ; Cells, Cultured ; Centrifugation/methods ; Histocytochemistry/instrumentation ; Histocytochemistry/methods ; Humans ; Polylysine/chemistry ; Reproducibility of Results ; Staining and Labeling/instrumentation ; Staining and Labeling/methods
    Chemical Substances Polylysine (25104-18-1)
    Language English
    Publishing date 2020-12-01
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot099622
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: Preparing Cell Smears for Staining.

    Rodig, Scott J

    Cold Spring Harbor protocols

    2020  Volume 2020, Issue 12

    Abstract: Increasing use is being made of cell smears for cell-staining studies. Suspension cells can be attached to slides by drying, and cell smears can also be prepared from biopsy samples, such as needle aspirates, tissue scrapings, or freshly dissected ... ...

    Abstract Increasing use is being made of cell smears for cell-staining studies. Suspension cells can be attached to slides by drying, and cell smears can also be prepared from biopsy samples, such as needle aspirates, tissue scrapings, or freshly dissected tissues. In these procedures, a thin layer of cells is deposited on a dry slide by physical methods. The most important factor in obtaining good staining patterns is that the smear be only a single cell thick. Tissue smears do not preserve tissue architecture, but are useful for identifying pathological changes and infectious organisms in tissue samples. Cell smears are easily prepared and can be fixed readily by any of the methods used for attached cells.
    MeSH term(s) Antibodies/metabolism ; Biopsy, Needle ; Histological Techniques/instrumentation ; Histological Techniques/methods ; Humans ; Immunohistochemistry/instrumentation ; Immunohistochemistry/methods ; Staining and Labeling/instrumentation ; Staining and Labeling/methods ; Tissue Fixation/instrumentation ; Tissue Fixation/methods
    Chemical Substances Antibodies
    Language English
    Publishing date 2020-12-01
    Publishing country United States
    Document type Journal Article
    ISSN 1559-6095
    ISSN (online) 1559-6095
    DOI 10.1101/pdb.prot099630
    Database MEDical Literature Analysis and Retrieval System OnLINE

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