Enhancement of immunochemical staining in aldehyde-fixed tissue

Abstract

A method for restoring immunoreactivity of a tissue, particularly decalcified tissue, fixed width an aldehyde fixative agent and embedded in an embedding medium, usually comprising celloidin, the method comprising the steps of contacting the tissue with an aldehyde releasing reagent solution comprising a solvent and an aldehyde releasing reagent and removing aldehyde released by the aldehyde releasing reagent from contact with the tissue by reacting the aldehyde in a substantially irreversible manner to form a non-aldehyde derivative, and removing excess base from the tissue. A preferred solution for celloidin-embedded decalcified tissue comprises methanolic sodium hydroxide at about one-third saturation.

1. A method for restoring immunoreactivity of a tissue fixed with an aldehyde fixing agent and embedded in an embedding medium, said method comprising the steps of:
 a) contacting said tissue with a solvent for said embedding medium and an aldehyde releasing reagent, which reagent releases aldehyde from said tissue by reacting said aldehyde in a substantially irreversible manner to form a non-aldehyde derivative; and  
 b) removing or neutralizing excess aldehyde releasing reagent from said tissue.  
 
     
     
       2. The method of  claim 1 , wherein said solvent is selected from the group consisting of methanol, ethanol, propanol, butanol, ethylene glycol, and glycerol. 
     
     
       3. The method of  claim 1 , wherein said aldehyde releasing reagent is selected from the group consisting of nucleophilic bases, oxidizing agents and organic acid/base pairs. 
     
     
       4. The method of  claim 1 , wherein said aldehyde releasing reagent is an oxidizing agent. 
     
     
       5. The method of  claim 1 , wherein said aldehyde releasing reagent is a nucleophilic base. 
     
     
       6. The method of  claim 1 , additionally comprising in step
 a) contacting said tissue with a chaotropic agent.  
 
     
     
       7. The method of  claim 1 , wherein said tissue comprises decalcified tissue. 
     
     
       8. A kit for use in immunostaining of a tissue fixed with an aldehyde fixing agent and embedded in an embedding medium, said kit comprising:
 a) a first container containing an aldehyde releasing reagent solution comprising an aldehyde releasing reagent selected from the group consisting of nucleophilic bases, oxidizing agents and organic acid/base pairs and a solvent for the aldehyde releasing reagent which is also capable of solubilizing the embedding medium; and    b) a second container containing (1) an immunostaining reagent or (2) a wash solution for removing excess aldehyde releasing reagent.    
     
     
       9. The method of claim    1     3  wherein said  solvent  aldehyde releasing reagent is an aqueous or organic solution. 
     
     
       10. A kit for use in immunostaining of a tissue fixed with an aldehyde fixing agent and embedded in an embedding medium, said kit comprising:
 a) a first container containing an aldehyde releasing reagent selected from the group consisting of nucleophilic bases, oxidizing agents and organic acid/base pairs;    b) a second container containing a solvent for solubilizing the embedding medium; and    c) a third container containing (1) an immunostaining reagent or (2) a wash solution for removing excess aldehyde releasing agents.    
     
     
       11. The kit of  claim 8  or  10  wherein said aldehyde releasing reagent comprises a nucleophilic base. 
     
     
       12. The kit of  claim 8  or  10  wherein said aldehyde releasing reagent comprises an oxidizing agent. 
     
     
       13. The kit of  claim 8  or  10  wherein said aldehyde releasing reagent comprises an organic acid/base pair. 
     
     
       14. The kit of  claim 8  or  10  wherein said solvent is selected from the group consisting of methanol, ethanol, propanol, butanol, ethylene glycol, and glycerol. 
     
     
       15. The kit of  claim 8  or  10  wherein said second container contains an immunostaining reagent comprising an antibody. 
     
     
       16. A method for restoring immunoreactivity of a tissue fixed with an aldehyde fixing agent and embedded in an embedding medium, said method comprising the steps of:
   a )  contacting said tissue with a solvent for said embedding medium and an organic acid/base pair aldehyde releasing reagent, which reagent releases aldehyde from said tissue by reacting said aldehyde in a substantially irreversible manner to form a non - aldehyde derivative; and        b )  removing or neutralizing excess aldehyde releasing reagent from said tissue.      
     
     
       17. The method of  claim 16 , wherein said solvent is selected from the group consisting of methanol, ethanol, propanol, butanol, ethylene glycol, and glycerol.  
     
     
       18. The method of  claim 16 , additionally comprising in step a)  contacting said tissue with a chaotropic agent.    
     
     
       19. The method of  claim 16 , wherein said tissue comprises decalcified tissue.  
     
     
       20. A method for restoring immunoreactivity of a tissue fixed with an aldehyde fixing agent and embedded in an embedding medium, said method comprising the steps of:
   contacting the tissue with a solvent for the embedding medium and an oxidizing reagent, wherein the reagent releases aldehyde from the tissue by reacting the aldehyde in a substantially irreversible manner to form a non - aldehyde derivative; and        removing or neutralizing excess reagent from the tissue.      
     
     
       21. The method of  claim 20 , wherein the solvent is selected from the group consisting of: methanol, ethanol, propanol, butanol, ethylene glycol, and glycerol.  
     
     
       22. The method of  claim 20 , further comprising contacting the tissue with a chaotropic agent.  
     
     
       23. The method of  claim 20 , wherein the tissue comprises decalcified tissue.  
     
     
       24. The method of  claim 20 , wherein the solvent is an aqueous or organic solution.