# US 8,465,944 B2 · Horizontal antigen retrieval > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,465,944 B2 - Document ID: US8465944B2 - Status: Granted - Kind: B2 - Application number: 12/526,323 - Filed: 2008-02-11 - Published: 2010-06-03 - Granted: 2013-06-18 - Earliest claimed: 2007-02-09 - Assignees: Dako Instrumec AS - Applicants: Nanna K. Christensen; Lars Winther - Inventors: Nanna K. Christensen; Lars Winther - Web page: https://patentgrep.com/patent/US8465944B2 ## CPC - G01N 33/50: Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing (measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q) - G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) 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. ### Claim 3 (depends on 1) The method according to claim 1, wherein the carrier is a glass slide, a slide made of a polymer material, or a membrane. ### Claim 4 (depends on 1) 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. ### Claim 5 (depends on 1) The method according to claim 1, wherein the target retrieval solution is heated to a temperature between 100° C. and 200° C. ### Claim 6 (depends on 1) The method according to claim 1, wherein the target retrieval solution is heated to a temperature between 100° C. and 150° C. ### Claim 7 (depends on 1) The method according to claim 1, wherein the target retrieval solution is heated to a temperature between 105° C. and 130° C. ### Claim 8 (depends on 1) 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. ### Claim 9 (depends on 1) 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. ### Claim 10 (depends on 1) 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. ### Claim 11 (depends on 10) The method according to claim 10, wherein the solvent is glycerol. ### Claim 12 (depends on 10) The method according to claim 10, wherein the solvent is selected from propylene glycol, ethylene glycol, and mixtures thereof. ### Claim 13 (depends on 1) 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. ### Claim 14 (depends on 1) The method according to claim 1, wherein the target retrieval solution comprises more than 25% (v/v) of a solvent. ### Claim 15 (depends on 1) The method according to claim 1, wherein the target retrieval solution comprises between 40% (v/v) and 80% (v/v) of a solvent. ### Claim 16 (depends on 1) The method according to claim 1, wherein the target retrieval solution comprises between 45% (v/v) and 75% of a solvent. ### Claim 17 (depends on 1) The method according to claim 1, wherein the target retrieval solution comprises about 50% (v/v) of a solvent. ### Claim 18 (depends on 1) 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. ### Claim 19 (depends on 1) The method according to claim 1, further comprising automation of one or more steps. ### Claim 20 (depends on 1) 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. ### Claim 21 (depends on 1) 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. ### Claim 22 (depends on 21) 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. ### Claim 23 (depends on 22) The method according to claim 22, wherein the further solvent is selected from water, an aqueous washing buffer, and ethanol. ### Claim 24 (depends on 21) The method according to claim 21, wherein the removing of the target retrieval is performed at ambient or above ambient temperature. ### Claim 25 (depends on 1) 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. ### Claim 26 (depends on 1) The method according to claim 1, further comprising staining the tissue or cell sample with a probe. ### Claim 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.