Methods and Compositions for Preparing Biological Specimens for Microscopic Analysis

Abstract

Methods and compositions are provided for preparing a biological specimen for microscopic analysis. These methods find many uses, for example in medicine and research, e.g., to diagnose or monitor disease or graft transplantation, to study healthy or diseased tissue, to screen candidate agents for toxicity and efficacy in disease modification. Also provided are reagents, devices, kits and systems thereof that find use in practicing the subject methods.

1 .- 53 . (canceled) 
     
     
         54 . A method for analysis of a subcellular biomolecule, comprising:
 cross-linking hydrogel subunits to biomolecules within a biological specimen by contacting the biological specimen with a fixation agent and a plurality of hydrogel subunits;   polymerizing the cross-linked hydrogel subunits to form a hydrogel-embedded biological specimen, thereby securing molecular components in place and/or maintaining the three-dimensional integrity of subcellular structures within the biological specimen;   labeling the hydrogel-embedded biological specimen by contacting it with a macromolecule that binds a subcellular biomolecule, wherein the macromolecule is: (i) conjugated to a detection moiety, (ii) bound to a secondary antibody conjugated to a detection moiety, or (iii) capable of being bound by the secondary antibody; and, if (iii), contacting the hydrogel-embedded biological specimen with the secondary antibody;   detecting the detection moiety, thereby performing three-dimensional analysis of the subcellular biomolecule within a biological specimen.   
     
     
         55 . The method of  claim 54 , further comprising permeabilizing the hydrogel-embedded biological specimen, thereby improving permeability of the specimen to the macromolecule. 
     
     
         56 . The method of  claim 54 , wherein the subcellular biomolecule is a protein or a nucleic acid. 
     
     
         57 . The method of  claim 56 , wherein the subcellular biomolecule is the protein, and the macromolecule is a polypeptide that specifically binds the subcellular protein. 
     
     
         58 . The method of  claim 57 , wherein the macromolecule is an antibody. 
     
     
         59 . The method of  claim 56  wherein the subcellular biomolecule is the nucleic acid, and the macromolecule is DNA or RNA that specifically hybridizes to a transcript of a gene of interest or to genomic material of interest. 
     
     
         60 . The method of  claim 59 , wherein the macromolecule comprises antisense RNA complementary to the transcript of the gene of interest. 
     
     
         61 . The method of  claim 59 , wherein the macromolecule comprises DNA complementary to the genomic material of interest. 
     
     
         62 . A method for maintaining the three-dimensional integrity of subcellular structures of a biological specimen while labeling subcellular biomolecules, comprising:
 fixing a biological specimen by contacting the biological specimen with a fixation agent and a plurality of hydrogel subunits, thereby cross-linking the hydrogel subunits to biomolecules within the biological specimen to produce biomolecule-bound hydrogel subunits;   polymerizing the biomolecule-bound hydrogel subunits to form a hydrogel-embedded biological specimen, thereby securing molecular components in place and maintaining the three-dimensional integrity of subcellular structures within the biological specimen;   contacting the hydrogel-embedded biological specimen with a macromolecule that specifically binds a subcellular biomolecule, thereby labeling the subcellular biomolecule.   
     
     
         63 . The method of  claim 62 , further comprising permeabilizing the hydrogel-embedded biological specimen, thereby improving permeability of the specimen to the macromolecule. 
     
     
         64 . The method of  claim 62 , wherein the subcellular biomolecule is a protein or a nucleic acid. 
     
     
         65 . The method of  claim 64 , wherein the subcellular biomolecule is the protein, and the macromolecule is a polypeptide that specifically binds the subcellular protein. 
     
     
         66 . The method of  claim 65 , wherein the polypeptide is conjugated to a detection moiety. 
     
     
         67 . The method of  claim 64 , wherein the subcellular biomolecule is the protein, and the macromolecule is an antibody, wherein the antibody is bound by a secondary antibody conjugated to a detection moiety. 
     
     
         68 . The method of  claim 64 , wherein the subcellular biomolecule is the nucleic acid, and the macromolecule is DNA or RNA that specifically hybridizes to a transcript of a gene of interest or genomic material of interest. 
     
     
         69 . The method of  claim 68 , wherein the macromolecule comprises antisense RNA complementary to the transcript of the gene of interest. 
     
     
         70 . The method of  claim 68 , wherein the macromolecule comprises DNA complementary to the genomic material of interest. 
     
     
         71 . A method for determining the distribution of a genetically encoded marker in a biological specimen at subcellular resolution, comprising:
 fixing a biological specimen by contacting the biological specimen with a fixation agent and a plurality of hydrogel subunits, thereby cross-linking the hydrogel subunits to biomolecules within the biological specimen to produce biomolecule-bound hydrogel subunits;   polymerizing the biomolecule-bound hydrogel subunits to form a hydrogel-embedded biological specimen, thereby securing molecular components in place within the biological specimen;   contacting the biological specimen with a macromolecule that specifically binds a genetically encoded marker, wherein the genetically encoded marker is a transcript of a gene of interest, and wherein the macromolecule comprises DNA or RNA that specifically hybridizes to the transcript and a detection moiety; and   detecting the detection moiety,   thereby determining the distribution of the genetically encoded marker in the biological specimen.   
     
     
         72 . The method of  claim 71 , wherein the macromolecule comprises antisense RNA complementary to the transcript of interest. 
     
     
         73 . The method of  claim 71 , wherein the macromolecule comprises DNA complementary to the genomic transcript of interest.