Methods and Compositions for Hematoxylin and Eosin Staining

Abstract

The present invention provide for solutions of a defined composition useful in a staining protocol, such as a hematoxylin and eosin staining protocol, when used at certain points of the staining protocol. The formulations of these defined solutions are such that carry-over of the solutions will not negatively impact, or preferably, will stabilize or favorably modify staining reagent solutions coming in contact with the solutions. In certain embodiments of the invention, solutions are buffered to maintain a specific pH that when carried-over—such as carried-over into hematoxylin—will not significantly influence the pH of the next staining reagent.

What is claimed is: 
     
         1 . A pH buffered solution comprising an organic acid and a polyhydroxy alcohol, wherein the pH buffered solution has a pH buffering range that comprises from about pH 2.0 to about pH 5.0. 
     
     
         2 . The pH buffered solution of  claim 1  wherein the pH buffered solution has a pH buffering range of from about pH 2.0 to about pH 5.0. 
     
     
         3 . The pH buffered solution of  claim 1  wherein the pH buffered solution comprises from about 5% to about 30% (v/v) of a polyhydroxy alcohol selected from the group consisting of ethylene glycol, propylene glycol, glycerin, and polyethylene glycol. 
     
     
         4 . The pH buffered solution of  claim 1  wherein the pH buffered solution comprises from about 5% to about 30% (v/v) of ethylene glycol. 
     
     
         5 . The pH buffered solution of  claim 1 , wherein the organic acid is selected from the group consisting of malic acid, citric acid, maleic acid, acetic acid, and tartaric acid. 
     
     
         6 . The pH buffered solution of  claim 1 , wherein the organic acid is selected from the group consisting of malic acid, citric acid, and tartaric acid. 
     
     
         7 . The pH buffered solution of  claim 1 , wherein the organic acid is tartaric acid. 
     
     
         8 . The pH buffered solution of  claim 1 , wherein the pH buffered solution comprises a concentration of from about 10 mM to about 100 mM of the organic acid. 
     
     
         9 . The pH buffered solution of  claim 1 , wherein the pH buffered solution is buffered such that the pH is maintained between about pH 2.0 to about pH 5.0 when water is introduced into the solution. 
     
     
         10 . The pH buffered solution of  claim 1  wherein the pH buffered solution comprises from about 10 mM to about 100 mM of tartaric acid and from about 5% to about 30% (v/v) of ethylene glycol. 
     
     
         11 . The pH buffered solution of  claim 1  further comprising an antimicrobial agent that is effective over at least a pH range of from about pH 2.0 to about pH 5.0. 
     
     
         12 . A solution with a pH of from about pH 2.0 to about pH 5.0 comprising aluminum salts and from about 5% to about 30% (v/v) of a polyhydroxy alcohol selected from the group consisting of ethylene glycol, propylene glycol, glycerin, and polyethylene glycol. 
     
     
         13 . The solution of  claim 12  wherein the aluminum salts are selected from the group comprising aluminum ammonium sulfate, aluminum sulfate, and aluminum potassium sulfate. 
     
     
         14 . A method of staining a tissue specimen with hematoxylin, the method comprising the steps of (i) contacting the specimen with a pH buffered solution of  claim 1  and (ii) transferring the specimen to a hematoxylin staining solution. 
     
     
         15 . The method of  claim 14  further comprising rinsing the specimen with water before contacting the specimen with the pH buffered solution. 
     
     
         16 . The method of  claim 15  wherein the pH buffered solution maintains a pH of from about pH 2.0 to about pH 5.0 when exposed to water carried over from prior contact of the specimen with water. 
     
     
         17 . The method of  claim 14  wherein the pH of the hematoxylin staining solution increases less than 0.30 pH units when exposed to pH buffered solution carried over from prior contact of the specimen with the pH buffered solution. 
     
     
         18 . The method of  claim 14  wherein the hematoxylin solution retains its staining capacity longer without an adverse change in staining characteristics than performing hematoxylin staining without contacting the specimen with the pH buffered solution. 
     
     
         19 . The method of  claim 14  wherein the method provides the capacity to stain at least about 2,700 standard specimen slides without an adverse change in staining characteristics using standard H & E staining procedures without changing the staining reagents. 
     
     
         20 . A kit for performing hematoxylin and eosin staining, the kit comprising a pH buffered solution of  claim 1  and at least one hematoxylin and eosin staining reagent selected from the group consisting of a hematoxylin solution, an eosin solution, a differentiating solution, and a bluing agent solution. 
     
     
         21 . The kit of  claim 20  comprising (i) a pH buffered solution of  claim 1 , (ii) a hematoxylin solution, (iii) an eosin solution, (iv) a differentiating solution, and (v) a bluing agent solution. 
     
     
         22 . The kit of  claim 20  wherein the pH buffered solution has a pH buffering range of from about pH 2.0 to about pH 5.0. 
     
     
         23 . The kit of  claim 20  wherein the pH buffered solution comprises from about 5% to about 30% (v/v) of a polyhydroxy alcohol selected from the group consisting of ethylene glycol, propylene glycol, glycerin, and polyethylene glycol. 
     
     
         24 . The kit of  claim 20  wherein the pH buffered solution comprises from about 5% to about 30% (v/v) of ethylene glycol. 
     
     
         25 . The kit of  claim 20 , wherein the organic acid is selected from the group consisting of malic acid, citric acid, maleic acid, acetic acid, and tartaric acid. 
     
     
         26 . The kit of  claim 20 , wherein the organic acid is selected from the group consisting of malic acid, citric acid, and tartaric acid. 
     
     
         27 . The kit of  claim 20 , wherein the organic acid is tartaric acid. 
     
     
         28 . The kit of  claim 20 , wherein the pH buffered solution comprises a concentration of from about 10 mM to about 100 mM of the organic acid. 
     
     
         29 . The kit of  claim 20 , wherein the pH buffered solution is buffered such that the pH is maintained between about pH 2.0 to about pH 5.0 when water is introduced into the solution. 
     
     
         30 . The kit of  claim 20  wherein the pH buffered solution comprises from about 10 mM to about 100 mM of tartaric acid and from about 5% to about 30% (v/v) of ethylene glycol.