Two phase immiscible system for the pretreatment of embedded biological samples
The present application provides a two phase immiscible system for the pretreatment of embedded biological samples comprising placing at least one support having an embedded biological sample on its surface into a pretreatment container, adding to the pretreatment container at least one reagent forming a layer, adding a carrier composition to the pretreatment container, such that reagent forming layer is formed on the top of the carrier composition, and in an amount such that the at least one reagent forming layer contacts at least a portion of the embedded biological sample. Pretreatment of the embedded biological samples can include removal of embedding medium from embedded biological, target retrieval and enzyme blocking samples before staining histochemical analysis or other processes. The system also includes an apparatus and processes of automation of the pretreatment methods.
1. A method for removing at least a portion of embedding medium from an embedded biological sample comprising:
placing at least one support having an embedded biological sample on its surface into a pretreatment container,
adding a carrier composition to the pretreatment container,
adding to the pretreatment container at least one reagent forming a layer on the surface of the carrier composition,
increasing the volume of carrier composition in the pretreatment container, at least until the at least one reagent forming layer contacts at least a portion of the embedded biological sample, and
removing at least a portion of the reagent forming layer by increasing the volume of the carrier composition in the pretreatment container.
2. The method according to claim 1 , further wherein the carrier composition is added to the pretreatment container after placing the at least one support into the pretreatment container, but before adding to the pretreatment container the at least one reagent forming a layer on the surface of the carrier composition, and further wherein said carrier composition does not contact the embedded biological sample.
3. The method according to claim 1 , further wherein the volume of the carrier composition is increased until the at least one reagent forming layer contacts the entirety of the embedded biological sample.
4. The method according to claim 1 , further wherein the two phase system is in constant motion whenever it is in contact with the biological sample.
5. The method according to claim 1 , further comprising removing at least a portion of the reagent forming layer by increasing the volume of the carrier composition in the pretreatment container until at least a portion of reagent forming layer overflows out of the pretreatment container.
6. The method according to claim 1 , further comprising removing at least a portion of the carrier composition from the pretreatment container, such that the reagent forming layer contacts at least a portion of the embedded biological sample a second time.
7. The method according to claim 1 , further comprising adding additional carrier composition to the pretreatment container therefore causing the reagent forming layer to contact at least a portion of a biological sample a third time followed by removing at least a portion of the carrier composition thus causing at least a portion of the reagent forming layer to contact at least a portion of the embedded biological sample a fourth time.
8. The method according to claim 1 wherein the reagent forming layer is at a temperature lower than the melting point of the embedding material.
9. The method according to claim 1 , wherein the embedding medium is selected from the group consisting of wax, paraffin, paramat, paraplats, peel away paraffin, tissue freezing medium, cryonic gel, embedding compound, polyester wax, and mixtures thereof.
10. The method according to claim 1 , wherein the reagent forming a layer comprises a solvent that is capable of dissolving the embedding medium.
11. The method according to claim 10 , wherein the reagent forming a layer is selected from the group consisting of hydrogenated naphthalene, naphthenic hydrocarbons, d-Limonenes, paraffinic/isoparaffinic hydrocarbons, paraffinic-glycol ether, an alkane hydrocarbon, and mixtures thereof.
12. The method according to claim 1 , wherein the carrier composition is an aqueous buffer solution capable of removing the liquefied embedding medium, and immiscible with the reagent forming layer.
13. The method according to claim 12 , wherein the carrier composition is selected from the group consisting of Tris-Buffered Saline Tween-20 (“TBST”), PBS, Hepes, MES buffer, traditional IHC target retrieval solutions, and DI water.
14. The method according to claim 1 , wherein the support is selected from a group consisting of a test tube, chip, array, disk and slide.
15. The method according to claim 1 , wherein the carrier composition is an aqueous buffer solution capable of removing the liquefied embedding medium, and immiscible with the reagent forming layer.
16. The method according to claim 15 , wherein the carrier composition is selected from the group consisting of Tris-Buffered Saline Tween-20 (“TBST”), PBS, Hepes, MES buffer, traditional IHC target retrieval solutions and DI water.
17. The method according to claim 1 , further comprising a rinsing steps after removal of the embedding medium with an alcohol or a diluted alcohol solution in water.
18. The method according to claim 17 , wherein the diluted alcohol is ethanol.
19. The method according to claim 18 , wherein the concentration of the ethanol solution is 30% ethanol or less.
20. The methods according to claim 1 further comprising staining of the pretreated biological samples.
21. The method according to claim 20 , further comprising a post staining clearing process.
22. The method according to claim 18 , wherein the post staining clearing process comprises exposing a stained biological sample or specimen to a solvent capable of removing embedding medium or a composition capable of removing solvent residues prior to cover slipping.