Patent ID: 8093012

Claim:
A method of detecting whether each of at least three targets are present or absent in a biological sample comprising the steps of: (a) performing antigen retrieval on the biological sample, wherein said antigen retrieval comprises the steps: i. de-paraffinize and re-hydrate biological sample and ii. boil in antigen retrieval buffer; (b) performing autofluorescence reduction on the biological sample prior to step (a), or step (c), wherein said autofluorescence reduction comprises incubating said biological sample in a solution comprising 1% hydrochloric acid and 70% ethanol; (c) contacting the biological sample with (1) a first antibody specific for a first target, (2) a first labeling reagent wherein said first labeling reagent comprises an antibody binding moiety for said first antibody and a first fluorescent detection moiety, (3) a second antibody specific for a second target, (4) a second labeling reagent comprising an antibody binding moiety for said second antibody and a second fluorescent detection moiety, (5) a third antibody specific for a third target, and (6) a third labeling reagent comprising an antibody binding moiety for said third antibody and a third fluorescent detection moiety; and (d) detecting a presence or an absence of each of said first, second and third labeling reagents, respectively, in said biological sample whereby the presence of said first, second or third labeling reagents indicates the presence of said first, second or third targets, respectively, in said biological sample and the absence of said first, second or third labeling reagents indicates the absence of said first, second or third targets, respectively, in said biological sample, wherein said biological sample is paraffin-embedded human tissue, and wherein prior to step (d) said sample is washed one or more times to remove unbound labeling reagent.