Deparaffinization compositions for dewaxing tissue specimens

Abstract

Compositions and methods are provided for dewaxing wax-embedded biological specimens prior to histochemical analysis. The compositions and methods provided can effectively remove wax or improved wax-based embedding materials, particularly paraffin-based, from specimens during preparation for histochemical or other diagnostic analyses, while minimizing danger to users, achieving compatibility with automated use, and maintaining compatibility with downstream histochemical analyses, particularly immunostaining. Compositions of the invention comprise a paraffin-solubilizing organic solvent, a polar organic solvent, and a surfactant. Compositions can further comprise water. The method involves contacting a wax-embedded specimen with the dewaxing composition to solubilize the wax impregnating the specimen prior to histochemical analysis. The method can comprise the further step of washing the dewaxed specimen immediately after dewaxing with an aqueous wash composition comprising a detergent to remove residual dewaxing composition. Also provided is a kit for dewaxing a wax-embedded specimen, which comprises a dewaxing composition and can further comprise a second composition of (1) an immunostaining reagent or (2) an aqueous wash solution comprising a detergent for removing residual dewaxing solution.

1 - 20 . (canceled)  
   
   
       21 . A dewaxing composition for removing wax from a wax-embedded immunoreactive biological tissue specimen comprising: 
 a) a paraffin-solubilizing organic solvent selected from the group consisting of aromatic hydrocarbons, terpenes and isoparaffinic hydrocarbons,    b) a polar organic solvent, and    c) a surfactant.    
   
   
       22 . The dewaxing composition of  claim 21  wherein said dewaxing composition comprises: 
 (a) approximately 25% to approximately 75% by volume of at least one paraffin-solubilizing organic solvent selected from the group consisting of aromatic hydrocarbons, terpenes and isoparaffinic hydrocarbons;    (b) approximately 25% to approximately 75% by volume of at least one water soluble polar organic solvent;    (c) approximately 0.5% to approximately 20% by weight to volume of at least one water soluble surfactant.    
   
   
       23 . The dewaxing composition of  claim 21 , which further comprises water at up to approximately 10% by volume of the composition.  
   
   
       24 . The dewaxing composition of  claim 21 , wherein the paraffin-solubilizing organic solvent is limonene.  
   
   
       25 . The dewaxing composition of  claim 21 , wherein the paraffin-solubilizing organic solvent comprises an isoparaffinic hydrocarbon.  
   
   
       26 . The dewaxing composition of  claim 21 , wherein the paraffin-solubilizing organic solvent is an alkylbenzene or dialkylbenzene.  
   
   
       27 . The dewaxing composition of  claim 21 , wherein the polar organic solvent is selected from the group consisting of C-1 to C-5 alcohols, C-3 to C-5 ketones, and C-2 to C-6 ethers.  
   
   
       28 . The dewaxing composition of  claim 27 , wherein the polar organic solvent comprises at least one solvent selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, tert-butanol, acetone, ethylene glycol, allyl alcohol, and propylene glycol.  
   
   
       29 . The dewaxing composition of  claim 21 , wherein the surfactant comprises a cationic or anionic surfactant.  
   
   
       30 . The dewaxing composition of  claim 29 , wherein the surfactant is a cationic surfactant having the formula  
     
       
         
         
             
             
         
       
     
     wherein R 1  is methyl, ethyl or propyl or isopropyl where n is 1 or 2; R 2  is an alkyl from C 8 H 17  to C 30 H 61  or a benzyl group; and R 3  is (CH 2 ) m  where m is from 1 to 10, or R 3  is (OCH 2 CH 2 ) p  where p is from 1 to 10.  
   
   
       31 . The dewaxing composition of  claim 30 , wherein the cationic surfactant is benzalkonium chloride or benzethonium chloride.  
   
   
       32 . The dewaxing composition of  claim 29 , wherein the surfactant is an anionic surfactant having the formula  
     
       
         
         
             
             
         
       
     
     wherein R 1  is C 6 H 13  to C 30 H 61  and R 3  is CH 2  or a phenyl group.  
   
   
       33 . The dewaxing composition of  claim 21 , wherein the surfactant comprises a non-ionic surfactant.  
   
   
       34 . The dewaxing composition of  claim 33 , wherein the non-ionic surfactant has the formula  
     
       
         
         
             
             
         
       
     
     wherein R is a linear or branched C1 to C10 alkyl group and X is from 5 to 40.  
   
   
       35 . The dewaxing composition of  claim 33 , wherein the non-ionic surfactant contains polyoxyethylene ethers of C 12  to C 20  fatty acids.