Quantifiable internal reference standards for immunohistochemistry and uses thereof

A method of determining the amount of a test antigen by immunohistochemistry (IHC), comprising:

• providing a formalin-fixed, paraffin-embedded (FFPE) cell or tissue sample comprising the test antigen and a quantifiable internal reference standard (QIRS) for the test antigen, the QIRS being a second antigen different from the test antigen, the FFPE sample having been prepared at a time T1 from an original cell or tissue sample having an original amount of the test antigen and an original amount of the QIRS;
• providing a reference calibration curve comprising at least a ratio that relates the amount of the test antigen to the amount of the QIRS in a reference FFPE sample at a time T2 after T1;
• measuring a first IHC signal corresponding to the QIRS in the FFPE sample at time T2, wherein the first IHC signal having a magnitude that varies in proportion to at least the concentration of the QIRS;
• measuring a second IHC signal corresponding to the test analyte in the FFPE sample at time T2, wherein the second IHC signal having a magnitude that varies in proportion to at least the concentration of the test analyte;
• and
• calculating the amount of the test antigen in the FFPE sample by applying the ratio(s) of the calibration curve as a conversion factor.