MULTIPLEX IMMUNOHISTOCHEMISTRY IMAGE CYTOMETRY
First Claim
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1. A method of performing immunohistochemistry on a formalin fixed paraffin embedded tissue section, the method comprising:
- contacting the section with a first tissue antigen specific antibody that specifically binds a tissue antigen wherein the first tissue antigen specific antibody optionally comprises a first enzyme label;
contacting the section with an enzyme-labeled antibody that specifically binds the first tissue antigen specific antibody if the first tissue antigen specific antibody does not comprise a first enzyme label;
contacting the section with a first colorimetric substrate of the first enzyme label, thereby visualizing one or more cells that express the first tissue antigen;
generating a first digital image of all or part of the section;
thereby completing a first staining cycle;
heating the section to at least 90°
C. for a sufficient time to remove the first tissue antigen specific antibody and the first labeled antibody from the one or more cells that express the first tissue specific antigen;
contacting the section with a second tissue antigen specific antibody that specifically binds a tissue antigen wherein the second tissue antigen specific antibody optionally comprises a second enzyme label;
contacting the section with an enzyme-labeled antibody that specifically binds the second tissue antigen specific antibody if the second tissue antigen specific antibody does not comprise a second enzyme label;
contacting the section with a second colorimetric substrate of the second enzyme label, and generating a second digital image of the section, thereby completing a second staining cycleprovided that the heating is performed between the first staining cycle and the second staining cycle.
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Abstract
Immunohistochemical (IHC) techniques that enable the sequential evaluation of at least seven biomarkers in one formalin-fixed paraffin-embedded (FFPE) tissue section are disclosed. The methods involve high-throughput multiplexed, quantitative IHC imaging, sequential IHC with iterative labeling, digital scanning, image coregistration and merging, and subsequent stripping of sections.
5 Citations
32 Claims
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1. A method of performing immunohistochemistry on a formalin fixed paraffin embedded tissue section, the method comprising:
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contacting the section with a first tissue antigen specific antibody that specifically binds a tissue antigen wherein the first tissue antigen specific antibody optionally comprises a first enzyme label; contacting the section with an enzyme-labeled antibody that specifically binds the first tissue antigen specific antibody if the first tissue antigen specific antibody does not comprise a first enzyme label; contacting the section with a first colorimetric substrate of the first enzyme label, thereby visualizing one or more cells that express the first tissue antigen; generating a first digital image of all or part of the section;
thereby completing a first staining cycle;heating the section to at least 90°
C. for a sufficient time to remove the first tissue antigen specific antibody and the first labeled antibody from the one or more cells that express the first tissue specific antigen;contacting the section with a second tissue antigen specific antibody that specifically binds a tissue antigen wherein the second tissue antigen specific antibody optionally comprises a second enzyme label; contacting the section with an enzyme-labeled antibody that specifically binds the second tissue antigen specific antibody if the second tissue antigen specific antibody does not comprise a second enzyme label; contacting the section with a second colorimetric substrate of the second enzyme label, and generating a second digital image of the section, thereby completing a second staining cycle provided that the heating is performed between the first staining cycle and the second staining cycle. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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Specification