7-Amino-4-methylcoumarin as a fluorescent substitute for Schiff’s reagent in periodic acid-Schiff staining: Its application to in-resin correlative light-electron microscopy

Periodic acid-Schiff (PAS) staining is useful for visualizing glycogen and mucus. 7-amino-4-methylcoumarin (AMC), an organic reagent, exhibits blue fluorescent signals upon UV excitation. We recently showed that AMC can be used as a fluorescent substitute for Schiff's reagent in PAS staining. In-resin correlative light-electron microscopy (CLEM) is an excellent technique employing fluorescent and electron microscopy (EM) images obtained from the same resin-embedded ultra-thin sections, and provides a high level of morphological concordance between the fluorescent and EM images. Here we studied whether AMC was useful in detecting PAS-positive cells in in-resin CLEM using human pathological samples. Formalin-fixed, paraffin-embedded sections of human colonic and renal tissues and colonic amoebiasis were used. After staining with AMC, these samples were subjected to EM preparation and embedded in epoxy resin. Semi-thin and ultra-thin sections were prepared, and fluorescent and EM images were obtained. AMC signals were well preserved in colonic goblet cells, renal basement membrane, and amoebic bodies in epoxy resin-embedded sections. Using the CLEM technique, a small number of amoebic bodies were easily detected in an inflammatory background of colonic amoebiasis. In conclusion, we successfully detected PAS-positive cells in in-resin CLEM with AMC. Our method may enhance EM analysis in human pathology.