Rapid fluorescent labeling of tissue for microdissection using fluorescent specific binding agents

Abstract

Methods are disclosed for rapid and specific fluorescent staining of biological tissue samples that substantially preserve biological molecules such as mRNA. Methods for microdissecting tissue to obtain pure populations of cells or tissue structures based upon identifying and excising cells or tissue structures that are labeled with fluorescent specific binding agents are also included. A laser capture microdissection apparatus useful for identifying and isolating cells and tissue structures following rapid immunofluorescent staining is also disclosed.

1 . A method of performing tissue microdissection of a tissue specimen, comprising: 
 exposing the tissue specimen to at least one fluorescently labeled specific binding agent that specifically binds to a component of interest in the tissue, wherein the tissue is exposed to a sufficient concentration of the agent, in an aqueous solution, for a sufficient period of time to label the component of interest without substantially degrading nucleic acids or proteins in the tissue;    washing unbound agent from the tissue, to produce a labeled tissue specimen;    intensifying an image of the labeled tissue specimen, to obtain an intensified fluorescent signal;    applying a transfer member to the labeled tissue specimen;    directing a target laser beam to the component of interest in the labeled tissue specimen, to mark the component that is to be dissected, while viewing the target laser beam through an infrared filter that selectively filters infrared radiation but not the fluorescent signal, to minimize heat distortion of the intensified image, while still viewing the intensified signal; and    applying radiant laser energy to the component of interest to transfer the component to the transfer member.    
     
     
         2 . The method of  claim 1 , wherein exposing the tissue to a sufficient concentration of the at least one fluorescently labeled specific binding agent comprises exposing the tissue to a concentration of at least 0.04 mg/mL of the at least one fluorescently labeled specific binding agent.  
     
     
         3 . The method of  claim 2 , wherein exposing the tissue to a sufficient concentration of the at least one fluorescently labeled specific binding agent comprises exposing the tissue to a concentration of at least 0.10 mg/mL of the at least one fluorescently labeled specific binding agent  
     
     
         4 . The method of  claim 1 , wherein exposing the tissue to the at least one fluorescently labeled specific binding agent comprises exposing the tissue to the at least one fluorescently labeled specific binding agent for less than about 5 minutes.  
     
     
         5 . The method of  claim 4 , wherein exposing the tissue to the at least one fluorescently labeled specific binding agent comprises exposing the tissue to the at least one fluorescently labeled specific binding agent for less than about 3 minutes.  
     
     
         6 . The method of  claim 5 , wherein exposing the tissue to the at least one fluorescently labeled specific binding agent comprises exposing the tissue to the at least one fluorescently labeled specific binding agent for no more than about 1 minute.  
     
     
         7 . The method of  claim 1 , wherein the aqueous solution comprises diethylpyrocarbonate-treated phosphate-buffered saline.  
     
     
         8 . The method of  claim 1 , wherein aqueous solution further comprises at least one enzyme inhibitor.  
     
     
         9 . The method of  claim 8 , wherein the enzyme inhibitor is an RNase inhibitor, a DNase inhibitor, a protease inhibitor, or a mixture of two or more thereof.  
     
     
         10 . The method of  claim 1 , wherein the at least one fluorescently labeled specific binding agent comprises: 
 a primary antibody covalently linked to a fluorescent molecule;    a premixed solution of primary antibody and secondary antibody covalently linked to a fluorescent molecule;    a premixed solution of a primary antibody covalently linked to a fluorescent molecule and a secondary antibody covalently linked to a fluorescent molecule;    a fluorescently labeled lectin;    a mixture of primary antibody and fluorescently labeled protein A or G; or    a mixture of primary antibody covalently linked to a fluorescent molecule and fluorescently labeled protein A or G.    
     
     
         11 . The method of  claim 1 , further comprising, prior to exposing the tissue specimen to at least one fluorescently labeled specific binding agent: 
 fixing the tissue specimen with a non-crosslinking fixative; and    rinsing the tissue specimen with an aqueous buffered solution for about 5 seconds per rinse;.    
     
     
         12 . The method of  claim 11 , wherein the non-crosslinking fixative comprises ethanol, acetone, methanol or a mixture of two or more thereof.  
     
     
         13 . The method of  claim 1 , wherein the component of interest comprises a cell expressing a particular antigen.  
     
     
         14 . The method of  claim 1 , wherein the tissue specimen is exposed to an aqueous environment for a combined time of less than about 5 minutes.  
     
     
         15 . The method of  claim 1 , further comprising, after applying radiant laser energy to the component of interest to transfer the component to the transfer member, recovering biomolecules from the component of interest.  
     
     
         16 . The method of  claim 16 , wherein the biomolecules comprise RNA, DNA, protein, or mixtures thereof.  
     
     
         17 . The method of  claim 15 , wherein the biomolecules are mRNA molecules.  
     
     
         18 . The method of  claim 16 , wherein the biomolecules are proteins, further comprising analyzing the proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE).  
     
     
         19 . A method of performing tissue microdissection of a tissue specimen, comprising: 
 exposing the tissue specimen to specific binding agent comprising a premixed solution of primary antibody and secondary antibody covalently linked to a fluorescent molecule, wherein the primary antibody specifically binds to a component of interest in the tissue, and wherein the tissue is exposed to at least 0.04 mg/mL of the primary antibody, in an aqueous solution, for a sufficient period of time to label the component of interest while preserving at least 20% of a target RNA in the tissue;    washing unbound specific binding agent from the tissue, to produce a labeled tissue specimen;    intensifying an image of the labeled tissue specimen, to obtain an intensified fluorescent signal;    applying a transfer member to the labeled tissue specimen;    directing a target laser beam to the component of interest in the labeled tissue specimen, to mark the component that is to be dissected, while viewing the target laser beam through an infrared filter that selectively filters infrared radiation but not the fluorescent signal, to minimize heat distortion of the intensified image, while still viewing the intensified signal; and    applying radiant laser energy to the component of interest to transfer the component to the transfer member.    
     
     
         20 . The method of  claim 19 , further comprising, after applying radiant laser energy to the component of interest to transfer the component to the transfer member, recovering nucleic acids and/or proteins from the component of interest.  
     
     
         21 . The method of  claim 1 , wherein the tissue specimen is frozen.