Abstract

 

Co-localizing two distinct antigens in formalin-fixed paraffin-embedded tissues via immunohistochemical techniques is challenging for light microscopy.  Two distinct antigens detected by two chromogen precipitates may mask each other under white light because darker chromogens obscure lighter colored ones.  As discovered by others, this can be overcome by use of a fluorescent chromogenic precipitate in combination with a DAB precipitate to co-localize two antigens.  We applied this technique to detect a subtype of tumor associated macrophages via co-localization of F4/80 and Arginase-1, of which the F4/80 antibody was functional only in immunohistochemistry but not immunofluorescence.  Imaging the fluorescent-labeled antigen in the red UV filter overcomes the masking problem due to the brown precipitate under white light.  Another limitation of detecting multiple colors under white light is that histochemical stains such as the hematoxylin and eosin (H&E) protocol routinely used in grading tumors excludes antigen detection via immunofluorescence.  The blue and red hues of the H&E stains are crucial for determining the extent of morphological dedifferentiation and thus provide most of the information.  We present evidence that a permanently fluorescent immunohistochemical chromogen can be incorporated into a histochemical stain such that the chromogen does not affect histochemical coloration, yet is co-localizable under UV light.  This  combination gives the pathologist the option of detecting a clinically relevant antigen within a histochemical stain and has the potential to improve tumor grading systems by providing better resolution regarding tumor subtypes.