STABILIZED TWO-PART HEMATOXYLIN SOLUTION UTILIZING pH ADJUSTMENT

Abstract

The present disclosure provides stabilized hematoxylin formulations having a pH of less than 2.4. The present disclosure also provides methods of using such stabilized hematoxylin formulations to stain biological samples.

1 . A stabilized hematoxylin formulation comprising hematoxylin dye and an acid in an amount ranging from about 0.2% to about 4% by total volume of the hematoxylin solution, and wherein the hematoxylin solution has a pH of less than 2.4, wherein the acid is selected from the group consist of hydrochloric acid, sulfuric acid, perchloric acid and nitric acid. 
     
     
         2 . The stabilized hematoxylin formulation of  claim 1 , wherein the pH is less than 2.1. 
     
     
         3 . The stabilized hematoxylin formulation of  claim 1 , further comprising a mordant, and an oxidant. 
     
     
         4 . The stabilized hematoxylin formulation of  claim 3 , further comprising a shelf-life extending agent. 
     
     
         5 . The stabilized hematoxylin formulation of  claim 4 , wherein the shelf-life extending agent is a polyol. 
     
     
         6 . The stabilized hematoxylin formulation of  claim 5 , wherein the polyol is selected from the group consisting of propylene glycol, ethylene glycol, and mixtures thereof. 
     
     
         7 . A method of staining a biological sample comprising: (a) increasing a pH of an aliquot of the stabilized hematoxylin formulation of  claim 1  to provide a hematoxylin staining solution comprising a pH ranging from about 2.4 to about 2.6, and (b) contacting the biological sample with the hematoxylin staining solution having the pH ranging from about 2.4 to about 2.6. 
     
     
         8 . The method of  claim 7 , wherein the pH of the aliquot of the stabilized hematoxylin formulation is increased by adding a readjustment solution comprising a strong base or a buffer. 
     
     
         9 . The method of  claim 8 , wherein the strong base is selected from the group consisting of sodium hydroxide, potassium hydroxide, lithium hydroxide, and ammonia. 
     
     
         10 . The method of  claim 8 , wherein the buffer has a pKa ranging from about 1.5 to about 3.5. 
     
     
         11 . The method of  claim 8 , wherein the readjustment solution comprises an additive selected from the group consisting of a shelf-life extending agent, an oxidant, and a mordant. 
     
     
         12 . The method of  claim 7 , wherein the pH of the stabilized hematoxylin formulation is increased prior to contacting the biological sample with the hematoxylin staining solution. 
     
     
         13 . A kit comprising a first component and a second component,
 the first component comprising a stabilized hematoxylin formulation comprising a hematoxylin dye and an acid in an amount ranging from about 0.1% to about 10% by total volume of the stabilized hematoxylin formulation, and wherein the stabilized hematoxylin formulation has a pH of less than 2.4; and   the second component comprising a strong base or a buffer, the second component provided in an amount relative to the first component such that when the first and second components are mixed, the pH of the hematoxylin solution increases to between about 2.4 to about 2.6.   
     
     
         14 . The kit of  claim 13 , wherein the first component further comprises a mordant, and an oxidant. 
     
     
         15 . The kit of  claim 13 , wherein the first component further comprises a shelf-life extending agent. 
     
     
         16 . The kit of  claim 15 , wherein the shelf-life extending agent is a polyol. 
     
     
         17 . The kit of  claim 16 , wherein the polyol is selected from the group consisting of propylene glycol, ethylene glycol, and mixtures thereof. 
     
     
         18 . The kit of  claim 13 , wherein the second component comprises a strong base selected from the group consisting of sodium hydroxide, potassium hydroxide and lithium hydroxide. 
     
     
         19 . The kit of  claim 13 , wherein the second component comprises a buffer selected from the group consisting of a cyanoacetate, fumarate, and sulfate. 
     
     
         20 . The kit of  claim 13 , further comprising a third component comprising a counterstain selected from the group consisting of eosin Y, orange G, light green SF yellowish, Bismark Brown, and fast green FCF.