The Feulgen reaction: from pink-magenta to rainbow fluorescent at the Maffo Vialli's School of Histochemistry

Abstract

For over a century, Palazzo Botta (Palace Botta) has housed the University of Pavia's Biomedical Institutes. Illustrious scientists have conducted research and taught at this Palace, making significant contributions to the advancement of natural, biological, and medical science. Among them, Camillo Golgi received the Nobel Prize for discovering the so-called "black reaction." Following Golgi, the Palace continued to be a hub for the development of methodologies and reactions aimed at detecting and quantifying biological components. Maffo Vialli (in the Golgi stream) was the first to establish a Histochemistry Research Group, which began in the naturalistic field and later expanded to the biomedical area. Among the many histochemical studies initiated in the Palace, the Feulgen reaction undoubtedly played a significant role. This reaction, developed R. Feulgen and H. Rossenbeck in 1924, had significant international implications: numerous researchers then contributed to define its fine chemical details, which remained the subject of study for years, resulting in a massive international scientific literature. The Pavia School of Histochemistry also contributed to the evolution and application of this method, which has become a true benchmark in quantitative histochemistry. Giovanni Prenna and the CNR Centre for Histochemistry made significant contributions, as they were already focused on fluorescence cytochemistry. The Pavia researchers made significant contributions to the development of methodology and, in particular, instrumentation; the evolution of the latter resulted in the emergence of flow cytometry and an ever-increasing family of fluorescent probes, which somewhat overshadowed the Feulgen reaction for DNA quantification. The advent of monoclonal antibodies then contributed to the final explosion of flow cytometry in clinical application, almost making young neophytes forget that its roots date back to Feulgen.

Figure 1.

The Botta Palace (1700) was originally the private residence of the family Botta-Adorno, and from 1890 to 2010, the location of the Biological Institutes of Pavia University (as it appears on top of the main entrance of the building: center panel). It currently houses the Golgi Museum (right panel) and the University's Kosmos Museum of Natural History.

Figure 2.

Professor Maffo Vialli and some of his historical collaborators attended a convivial event in the Comparative Anatomy Institute's library, in the 1970s.

Figure 3.

Different models of home-made histophotometers by Vialli’s group.

Figure 4.

The first commercial Leitz MPV1 cytophotometer and (right) micrographs of frog erithrocyte nuclei that were Feulgenlabelled with different Shiff-type reagents.

Figure 5.

Some of the chemical drugs tested by Giovanni Prenna as possible fluorescent Shiff-type reagents and examples of Feulgenstained HeLa cell nuclei.

Figure 6.

Multicolor fluorescent examples of rainbow labeling. DHD cells: a) supravital single labelling with Hoechst 33342; b) supravital dual-labelling with Hoechst 33342 and propidium iodide; c) apoptotic HeLa cells after supravital dual-labelling with FITC-conjugated Annexin V and propidium iodide.

Figure 7.

The advent of monoclonal antibodies. a) BrdU-labeled HeLa cells revealed by a FITC-conjugated anti-BrdU antibody, and b) counterstained with propidium iodide; c) merged image..