Haematoxylin

The spheroids were fixed with Paraformaldehyde 4% (Sigma-Aldrich, St. Louis, MO, USA) and embedded in paraffin. The paraffin sections were rehydrated by using both xylene and a descending-graded series of ethyl alcohol. After washing with the 50% alcohol, the sections were kept for five minutes in distilled water. Then, they were stained with haematoxylin (Bio-Optica, Milan, Italy) for 2 min, washed in distilled water, and then stained with Eosin (Bio-Optica) for 2 min; subsequently, they were washed in distilled water. Finally, they were dehydrated through an ascending graded series of ethyl alcohol and xylene and were mounted with Eukitt Solution (Orsatec GmbH, Kindler GmbH and Co., Bobingen, Germany).

Chemicals and reagents used for metabolite extraction were all HPLC grade. Water was treated in a Milli-Q water purification system (Millipore, Bedford, MA, USA) before use. The standards used for the identification of intracellular HF metabolites were from Sigma Chemical Co. (St. Louis, MO, USA). Haematoxylin and Eosin were provided by Bio-Optica (Milan, Italy). MitoTracker Red CMXRos (M7512, Invitrogen, Carlsbad, CA, USA) used for ex vivo staining of mitochondria in skin biopsies was reconstituted in DMSO and 1 mM stock aliquots were stored at −20 °C before use.