Methods and systems for quantitative immunohistochemistry

A method of amplifying a signal for a target protein biomarker in a sample, said method comprising:

• a. applying to the sample a biomarker-specific agent specific for the target protein biomarker;
• b. applying to the sample a second binding agent specific for the biomarker-specific agent, the second binding agent is conjugated with a secondary enzyme;
• c. applying to the sample a tyramide agent comprising a tyramide molecule conjugated with a tyramide hapten, wherein the secondary enzyme catalyzes deposition of the tyramide hapten onto the sample at a location of the second binding agent;
• d. applying to the sample a third binding agent specific for the tyramide hapten, the third binding agent is conjugated with a tertiary enzyme;
• and
• e. applying to the sample a detectable moiety, wherein the tertiary enzyme catalyzes a reaction with the detectable moiety to make the detectable moiety visible;
• whereby the detectable moiety is visible as a punctate dot using brightfield microscopy, wherein the punctate dot is indicative of the target protein biomarker.