Representative example of quantification of Acr-dG immunofluorescence using
MetaMorph. For immunofluorescence analysis of Acr-dG, formalin-fixed paraffin-embedded
(FFPE) oral cells were subjected to a standard procedure using a monoclonal antibody
against Acr-dG. Cy3 was used as the secondary antibody, and nuclei were counterstained
with 4′,6-diamidino-2-phenylindole (DAPI). Samples were imaged using a HistoRx PM-2000
imaging device, and images were analyzed to identify and enumerate positively stained
nuclei using MetaMorph software. (A) Original image in Cy3 channel staining for
Acr-dG. (B) Cy3 image; green nuclei are those identified “positive for Cy3” by
MetaMorph. (C) Original image in DAPI channel. (D) DAPI image-integrated morphometry
mask to eliminate large and small objects. (E) Resulting filtered DAPI image. (F)
Final DAPI image; green nuclei are those identified “positive for Cy3” by MetaMorph,
and red nuclei are those identified “negative for Cy3” by MetaMorph. The scale bar
size shown is 100 µm.