Method for immunohistochemical detection of collagen in a tissue sample
A technique of tissue samples and immunohistochemistry, which is applied in the direction of measuring devices, biological tests, material inspection products, etc., can solve the problems of diffusion, ambiguity of tissue shape, and unrecognizable cell structure.
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[0020] All publications cited herein are hereby incorporated by reference. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[0021] The terms "comprising", "comprising", "containing", and "having" are used herein in their open, non-limiting sense.
[0022] An "antibody" as used herein refers to an immunoglobulin molecule or an immunologically active portion of an immunoglobulin molecule that has a specific amino acid sequence and binds only an antigen or group of closely related antigens. Examples of "antibodies" include IgG, IgM, IgA, IgD and IgE. Examples of immunologically active portions of immunoglobulin molecules include Fab and F(ab)', which can be produced by treating antibodies with enzymes, such as pepsin 2 fragment. In particular, an "antibody" as used herein binds one or more types of collagen, but does not substantially bind...
Embodiment 1
[0054] Methods and materials
[0055] Formaldehyde-fixed, paraffin-embedded human checkered tissue blocks (Dako, Carpenturia, CA; Biomeda, Foster City, CA) were routinely processed for immunohistochemistry (D'Andrea et al., 2003; Neuroscience Letters 333(3):163-166). Tissues measured in this study were brain (n=10), adrenal gland (n=10), colon (n=6), small intestine (n=2), stomach (n=2), heart (n=6), liver (n=10), skin (n=3), kidney (n=8), lung (n=10), pancreas (n=10), testis (n=8), ovary (n=8), prostate (n=8), n=8), uterus (n=8), thyroid (n=10) and spleen (n=10). Tissue sections on microscopic slides were deparaffinized and rehydrated before use according to conventional methods (D'Andrea et al., 2003, supra). The various collagenases used in this study were bacterial collagenases, Clostripain A types IA, IV, and XI (Sigma, St. Louis. MO, product numbers C0130, C5138, and C7657) in collagenase buffer solution (containing 11.47g / l TES free acid (Sigma, product number T-137...
Embodiment 2
[0059] Enhancement of Immunohistochemical Detection of Collagen IV in Formaldehyde-Fixed, Paraffin-Embedded Normal Human Brain Tissue by Enzymatic Pretreatment
[0060] To compare the effects of various pretreatment methods on the immunohistochemical detection of collagen in formaldehyde-fixed, paraffin-embedded normal human brain tissue. Similar experiments can be performed to compare the effect of pretreatment methods on other types of tissue samples, such as fresh tissue sections, frozen tissue sections, or other types of fixed and embedded tissues.
[0061] Blocks of normal human brain tissue in a checkered pattern (Dako, Carpenturia, CA; Biomeda, Foster City, CA) were used for this study. The tissue is heated, passed a general protease (general protease), such as pepsin (pre-diluted from the supplier), trypsin (1mg / ml) or proteinase K (1mg / ml), or collagenase, clostripidin Enzyme A type IV (1 mg / ml), pretreated using the procedure described in Example 1. Immunohistochem...
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