A method of detecting multiple antigens from a formalin-fixed paraffin-embedded tissue sample comprising: (a) subjecting the sample to an antigen retrieval process to expose one or more antigens in the sample, wherein the antigen retrieval process is performed at a specific pH6 or pH9 for the antigen being retrieved; (b) applying a hydrogen peroxide to block endogenous detection of peroxidase (c) applying a second blocking reagent to block against nonspecific binding of one or more antigens; (d) incubating the sample with an antibody that binds to one target in the sample; (e) incubating the sample with a secondary antibody bound to HRP; (f) incubating the sample with 3-amino-9-ethylcarbazole (AEC); (g) detecting a signal from the AEC; (h) removing the AEC or destaining the signal from step (d) by sequentially: immersing the sample in an organic solvent-based destaining buffer comprising 50% ethanol for 2 mins, immersing the sample in an organic solvent-based destaining buffer comprising 90% ethanol for 5 mins, and immersing the sample in an organic solvent-based destaining buffer comprising 50% ethanol for 2 mins, wherein the removal of AEC or destaining does not remove the antibody and allows for the preservation of tissue antigenicity and tissue architecture of the sample and the repeated performance of every step on the sample; (i) repeating steps (a) through (h) at least one time, and wherein when steps (a) through (h) are repeated, the method comprises a further step of applying a third blocking reagent to block against specific binding of the antibody used previously in step (d). (a) subjecting the sample to an antigen retrieval process to expose one or more antigens in the sample, wherein the antigen retrieval process is performed at a specific pH6 or pH9 for the antigen being retrieved; (b) applying a hydrogen peroxide to block endogenous detection of peroxidase (c) applying a second blocking reagent to block against nonspecific binding of one or more antigens; (d) incubating the sample with an antibody that binds to one target in the sample; (e) incubating the sample with a secondary antibody bound to HRP; (f) incubating the sample with 3-amino-9-ethylcarbazole (AEC); (g) detecting a signal from the AEC; (h) removing the AEC or destaining the signal from step (d) by sequentially: immersing the sample in an organic solvent-based destaining buffer comprising 50% ethanol for 2 mins, immersing the sample in an organic solvent-based destaining buffer comprising 90% ethanol for 5 mins, and immersing the sample in an organic solvent-based destaining buffer comprising 50% ethanol for 2 mins, wherein the removal of AEC or destaining does not remove the antibody and allows for the preservation of tissue antigenicity and tissue architecture of the sample and the repeated performance of every step on the sample; immersing the sample in an organic solvent-based destaining buffer comprising 50% ethanol for 2 mins, immersing the sample in an organic solvent-based destaining buffer comprising 90% ethanol for 5 mins, and immersing the sample in an organic solvent-based destaining buffer comprising 50% ethanol for 2 mins, wherein the removal of AEC or destaining does not remove the antibody and allows for the preservation of tissue antigenicity and tissue architecture of the sample and the repeated performance of every step on the sample; (i) repeating steps (a) through (h) at least one time, and wherein when steps (a) through (h) are repeated, the method comprises a further step of applying a third blocking reagent to block against specific binding of the antibody used previously in step (d). <EOS>