A generally applicable sequential alkaline phosphatase immunohistochemical double staining

A universal type of sequential double alkaline phosphatase immunohistochemical staining is described that can be used for formalin-fixed, paraffin-embedded and cryostat tissue sections from human and mouse origin. It consists of two alkaline phosphatase detection systems including enzymatic visualizations in red and blue. Both red and blue enzymatic reaction products did not shield the first set of reagents from preventing cross reaction with the second staining sequence. However, cross reaction between the two staining sequences was fully abolished by a heat-induced epitope retrieval step, applied for 10 min at 98 degrees C. This step removed primary and secondary antibodies effectively but did not affect the quality of the enzymatic reaction products.