Horizontal antigen retrieval
View Patent ↗The present invention relates to a method for enhancing immunoreactivity of a tissue or cell sample fixed in a fixing medium, a target retrieval composition and its use. The method comprises providing a carrier in a horizontal position, said carrier having thereon a tissue or cell sample, said tissue or cell sample being on top of the carrier; contacting substantially the tissue or cell sample side of the carrier with a buffered target retrieval solution, wherein the target retrieval solution remains otherwise exposed to the environment; heating the tissue or cell sample and the target retrieval solution to a temperature above 100° C. The invention furthermore concerns the automated immunohistochemical staining of said samples, in particular the reactivation of antigens masked by fixation.
1. A method for enhancing immunoreactivity of a tissue or cell sample fixed in a fixing medium, comprising:
providing a carrier in a horizontal position, said carrier having thereon a tissue or cell sample, said tissue or cell sample being on top of the carrier;
applying an amount of a buffered target retrieval solution to the tissue or cell sample side of the carrier to cover the tissue or cell sample without submerging the carrier, wherein the target retrieval solution remains otherwise exposed to the environment; and
heating the tissue or cell sample and the target retrieval solution at a temperature above 100° C.
2. The method according to claim 1 , wherein more than one tissue or cell sample is placed on top of the carrier, and where in the amount of buffered target retrieval solution applied covers the more than one tissue or cell sample.
3. The method according to claim 1 , wherein the carrier is a glass slide, a slide made of a polymer material, or a membrane.
4. The method according to claim 1 , wherein the buffered target retrieval solution does not evaporate to a measurable degree when heated at a temperature between 100° C. and 150° C.
5. The method according to claim 1 , wherein the target retrieval solution is heated to a temperature between 100° C. and 200° C.
6. The method according to claim 1 , wherein the target retrieval solution is heated to a temperature between 100° C. and 150° C.
7. The method according to claim 1 , wherein the target retrieval solution is heated to a temperature between 105° C. and 130° C.
8. The method according to claim 1 , wherein the target retrieval solution comprises a solvent having a boiling point significantly higher than the boiling point of water.
9. The method according to claim 1 , wherein the target retrieval solution comprises a solvent having a boiling point higher than the boiling point of water and lower than 199° C.
10. The method according to claim 1 , wherein a solvent of the target retrieval solution is selected from the group consisting of alcohols, glycols, ketones, esters, amides, nitriles, and mixtures thereof.
11. The method according to claim 10 , wherein the solvent is glycerol.
12. The method according to claim 10 , wherein the solvent is selected from propylene glycol, ethylene glycol, and mixtures thereof.
13. The method according to claim 1 , wherein the target retrieval solution further comprises one or more reagents capable of breaking bonds formed during fixation of the biological sample.
14. The method according to claim 1 , wherein the target retrieval solution comprises more than 25% (v/v) of a solvent.
15. The method according to claim 1 , wherein the target retrieval solution comprises between 40% (v/v) and 80% (v/v) of a solvent.
16. The method according to claim 1 , wherein the target retrieval solution comprises between 45% (v/v) and 75% of a solvent.
17. The method according to claim 1 , wherein the target retrieval solution comprises about 50% (v/v) of a solvent.
18. The method according to claim 1 , wherein the fixing medium is selected from formaldehyde, Bouins solution, Periodate/Lysine/Paraformaldehyde, paraformaldehyde, Boonfix I, Boonfix II, Myrsky's fixative, glutaraldehyde, a zinc formalin, an aldehyde, and mixtures thereof.
19. The method according to claim 1 , further comprising automation of one or more steps.
20. The method according to claim 1 , further comprising cooling the carrier and the target retrieval solution after heating the tissue or cell sample and the target retrieval solution.
21. The method according to claim 1 , further comprising removing the target retrieval solution from the surface of the carrier after (i) heating the tissue or cell sample and the target retrieval solution and/or (ii) cooling the carrier and the target retrieval solution.
22. The method according to claim 21 , wherein the target retrieval solution is removed from the surface of the carrier using a further solvent capable of mixing with the target retrieval solution.
23. The method according to claim 22 , wherein the further solvent is selected from water, an aqueous washing buffer, and ethanol.
24. The method according to claim 21 , wherein the removing of the target retrieval is performed at ambient or above ambient temperature.
25. The method according to claim 1 , further comprising renewing or replacing the target retrieval solution one or more times during the heating of the tissue or cell sample and the target retrieval solution.
26. The method according to claim 1 , further comprising staining the tissue or cell sample with a probe.
27. A method for enhancing the immunoreactivity of a tissue or cell sample fixed in a fixing medium, wherein the method comprises applying an amount of a target retrieval solution to the tissue or cell sample to cover the tissue or cell sample without submerging the carrier; and wherein the target retrieval solution comprises glycerol, water, and one or more further reagents selected from a nucleophile, a buffer, a chelator, and a detergent.