Tissue staining method, tissue evaluation method and biosubstance detection method

Abstract

A tissue staining method which comprises: staining a tissue with a staining reagent wherein a biosubstance recognition site is bonded to particles carrying multiple fluorescent substances accumulated therein; in the stained tissue, counting fluorescent points or measuring fluorescent brightness; and evaluating the expression level of a biosubstance, which matches the biosubstance recognition site, in the aforesaid tissue on the basis of the number of the fluorescent points or fluorescent brightness that was measured.

1 . A method for quantification of target substance (s) present in a stained tissue, comprising:
 counting a number of bright spot (s) representing fluorescence emitted from particles bound to the target substance(s) present in the stained tissue;   quantifying the target substance (s) present in the stained tissue on the basis of the counted number of the bright spot (s) representing the fluorescence emitted from the particles bound to the target substance(s) present in the stained tissue,   wherein the stained tissue has been stained with a staining reagent which comprises the particles,   each of the particles comprises (i) a fluorescent dye dispersed therein, held therein, or held thereon, and (ii) a target-substance-recognizing body bound to the particle, and   the particles are bound to the target substance (s) present in the stained tissue via the target-substance-recognizing body.   
     
     
         2 . The tissue evaluation method of  claim 1 , wherein the particles comprising the fluorescent dye and bound to the target substance-recognizing body have an average particle diameter in the range of 40 to 500 nm. 
     
     
         3 . The tissue evaluation method of  claim 1 , wherein the particles have a variation coefficient of particle diameter size of 20% or less. 
     
     
         4 . The tissue evaluation method of  claim 1 , further comprising:
 measuring brightness of each bright spot in the stained tissue;   determining a brightness distribution on the basis of the number of the counted bright spots and the brightness of each bright spot;   calculating the brightness per each of the fluorescent dye-containing particles on the basis of the brightness distribution;   calculating the number of the fluorescent dye-containing particles bound to the stained tissue on the basis of the sum of the measured brightness of the bright spots and the calculated brightness per each of the fluorescent dye-containing particles; and   determining the expression level of the one or more target substance (s) in the stained tissue on the basis of the calculated number of the fluorescent dye-containing particles.   
     
     
         5 . The tissue evaluation method of  claim 1 , wherein the fluorescent dye-containing particles comprises:
 a first set of fluorescent dye-containing particles bound to a first set of target-substance-recognizing bodies; and   a second set of fluorescent dye-containing particles bound to a second set of target-substance-recognizing bodies;   wherein the second set of fluorescent dye-containing particles have a fluorescence wavelength different from the first set of fluorescent dye-containing particles; and   wherein the second set of target-substance-recognizing bodies is different from the first set of target-substance-recognizing bodies.   
     
     
         6 . The tissue evaluation method of  claim 5 , wherein the first set of target-substance-recognizing bodies comprises an anti-human estrogen receptor (ER) antibodies, and the second set of target-substance-recognizing bodies comprise anti-human epidermal growth factor receptor 2 (HER2) antibodies. 
     
     
         7 . The tissue evaluation method of  claim 1 , further comprising:
 determining a brightness per fluorescent dye-containing particle based on a brightness distribution of the bright spots in the stained tissue; and   determining a total number of the fluorescent dye-containing particles per cell in the stained tissue by dividing a total brightness per cell by the brightness per fluorescent dye-containing particle.   
     
     
         8 . The tissue evaluation method of  claim 1 , wherein the one or more target substance(s) are proteins and the target-substance-recognizing bodies bind to the proteins. 
     
     
         9 . The tissue evaluation method of  claim 8 , wherein the target-substance-recognizing bodies comprise anti-human estrogen receptor (ER) antibodies or anti-human epidermal growth factor receptor 2 (HER2) antibodies. 
     
     
         10 . The tissue evaluation method of  claim 1 , wherein the particles are silica nanoparticles, polystyrene nanoparticles, or polylactic acid nanoparticles. 
     
     
         11 . The tissue evaluation method of  claim 1 , wherein the particles are silica nanoparticles. 
     
     
         12 . The tissue evaluation method of  claim 1 , wherein the fluorescent dye comprises at least one dye selected from a group consisting of fluorescein dye molecules, rhodamine dye molecules, green dye molecules, cascade dye molecules, coumarin dye molecules, eosin dye molecules, nitrobenzoxadiazole (NBD) dye molecules, pyrene dye molecules, red dye molecules, cyanine dye molecules and combinations thereof. 
     
     
         13 . The tissue evaluation method of  claim 1 , wherein the fluorescent dye has an excitation wavelength of 200-700 nm and an emission wavelength of 400-900 nm. 
     
     
         14 . The tissue evaluation method of  claim 13 , wherein the excitation wavelength is from 550-650 nm and the emission wavelength is from 570-670 nm. 
     
     
         15 . The tissue evaluation method of  claim 1 , wherein the particles further comprise quantum dots dispersed therein, held therein, or held thereon. 
     
     
         16 . The tissue evaluation method of  claim 3 , wherein the variation coefficient is in the range of 5 to 15%. 
     
     
         17 . The tissue evaluation method of  claim 2 , wherein the average particle diameter is 40 to 204 nm.