Methods and compositions for a microemulsion-based tissue treatment

Abstract

The invention is directed to methods and compositions for deparaffinizing paraffin-embedded biological samples for subsequent tissue staining. The compositions are microemulsions that may include water/oil/surfactant microemulsions, and optionally a cosurfactant. The microemulsions enable deparaffinization without the use of xylene or toluene, and also enable solvent exchange without the use of intermediary alcohol dehydration or alcohol rehydration compositions.

1 . A method of removing paraffin-based embedding medium from a paraffin-embedded biological sample comprising: 
 contacting the paraffin-embedded biological sample with a deparaffinizing microemulsion comprising surfactant, nonpolar organic solvent and water, wherein the surfactant is soluble in both the water and the nonpolar organic solvent, thereby transferring the paraffin to the microemulsion; and    removing the microemulsion.    
     
     
         2 . The method of  claim 1  wherein the surfactant is a non-ionic surfactant.  
     
     
         3 . The method of  claim 1  wherein the surfactant is selected from the group consisting essentially of ethoxylated alkyl alcohols and ethoxylated alkyl carboxylic acids.  
     
     
         4 . The method of  claim 1  wherein removing the microemulsion comprises rinsing it away.  
     
     
         5 . The surfactant of  claim 1  having the following structural formula: 
         R 1—X—(CH   2 CH 2 O) n —R 2   
       wherein R1 is a long-chain linear or branched alkane hydrocarbon from about C5 to about C30; X is a linking group comprising an ether, ester, carbonate, benzyl, or sorbitol; n is from about 5 to about 20, and R2 is a hydrogen.  
     
     
         6 . The method of  claim 1  wherein the surfactant is selected from the group consisting of Tomadol 1-5, Tomadol 91-6, Tomadol 1-7, Tomadol 23-6.5, Tomadol 91-8, Tomadol 1-73B; Tomadol 900; polyethylene glycol 400 laurate; Tergitol 15-S-7, Tergitol 15-S-9, and hexaethylene glycol tridecane ether.  
     
     
         7 . The method of  claim 1  wherein said nonpolar organic solvent is selected from the group consisting of terpenes, alkylbenzenes, aromatic solvents, normal paraffin oils and branched paraffin oils.  
     
     
         8 . The method of  claim 1  wherein the nonpolar organic solvent comprises LIQUID COVERSLIP.  
     
     
         9 . The method of  claim 1  wherein the nonpolar organic solvent comprises NORPAR 15.  
     
     
         10 . The method of  claim 1  wherein the deparaffinizing microemulsion has a water to surfactant range from about 0:1 to about 10:1 by weight.  
     
     
         11 . The method of  claim 1  wherein the deparaffinizing microemulsion has a water to nonpolar organic solvent range from about 0:1 to about 10:1 by weight.  
     
     
         12 . The method of  claim 1  wherein the deparaffinizing microemulsion comprises surfactant at a weight percentage from about 5% to about 90%, oil at a weight percentage from about 5% to about 90%, and water at a weight percentage from 0% to about 90%.  
     
     
         13 . A method of preparing a paraffin-embedded biological sample for staining comprising: 
 deparaffinizing the paraffin-embedded biological sample by dissolving the paraffin with a nonpolar organic solvent; and    exchanging the nonpolar organic solvent in the deparaffinized tissue with an exchanging composition comprising surfactant and water, wherein the surfactant is soluble in both the water and the nonpolar organic solvent.    
     
     
         14 . The method of  claim 13  wherein the exchanging composition comprises Tomadol 1-73B:water, and Tergitol 15-S-7:water at a weight ratio of 1:4, respectively.  
     
     
         15 . The method of  claim 13  wherein the deparaffinizing step optionally includes heating the sample.  
     
     
         16 . A method of preparing a paraffin-embedded biological sample for staining comprising: 
 deparaffinizing the paraffin-embedded biological sample by dissolving the paraffin with a nonpolar organic solvent; and    exchanging the nonpolar organic solvent in the deparaffinized tissue with a microemulsion comprising surfactant, nonpolar organic solvent and water wherein the surfactant is soluble in both the water and the nonpolar organic solvent.    
     
     
         17 . A method of preparing a paraffin-embedded biological sample for staining comprising: 
 deparaffinizing the paraffin-embedded biological sample by dissolving the paraffin with a nonpolar organic solvent; and    exchanging the nonpolar organic solvent in the deparaffinized tissue with a microemulsion comprising surfactant, nonpolar organic solvent, water, and polar organic cosurfactant wherein the surfactant is soluble in both the water and the nonpolar organic solvent.    
     
     
         18 . The method of  claim 17  wherein the polar organic cosurfactant is selected from the group consisting of C-1 to C-5 alcohols, C-3 to C-5 ketones, C-2 to C-6 ethers, and C-2 to C-8 diols.  
     
     
         19 . The method of  claim 17 , wherein the polar organic cosurfactant comprises a solvent or a mixture of solvents selected from the group consisting of methanol, ethanol, isopropanol, butanol, acetone, ethylene glycol, propylene glycol, tert butanol, pentanediol, hexanediol, and octanediol.  
     
     
         20 . A method of preparing a deparaffinized tissue sample containing a nonpolar organic solvent for subsequent staining, comprising contacting said tissue with a microemulsion comprising a surfactant, a nonpolar organic solvent and water for enough time to replace the nonpolar organic solvent with the microemulsion wherein the surfactant is soluble in both the water and the nonpolar organic solvent.  
     
     
         21 . A method of exchanging water-for-oil in a tissue sample predominantly containing water comprising contacting the tissue sample with an exchanging composition comprising a surfactant in oil wherein the surfactant is also soluble in water.  
     
     
         22 . A method of exchanging oil-for-water in a tissue sample predominantly containing oil comprising contacting the tissue sample with an exchanging composition comprising a surfactant in oil wherein the surfactant is also soluble in water.  
     
     
         23 . A method of exchanging oil-for-water in a tissue sample predominantly containing oil comprising contacting the tissue sample with an exchanging composition comprising a surfactant in water wherein the surfactant is also soluble in oil.  
     
     
         24 . A method of exchanging water-for-oil in a tissue sample predominantly containing water comprising contacting the tissue sample with an exchanging composition comprising a surfactant in water wherein the surfactant is also soluble in oil.