Periodic acid

In kidneys, scheduled for histopathology, dehydration and embedding in paraffin was performed. Kidney tissue was cut and sections were transferred on glass slides. To assess glomerulosclerosis periodic acid–Schiff (PAS) staining was performed. Briefly, sections were deparaffinized in Xylene and subsequently rehydrated in a descending ethanol series. After incubation for 10 min each in 0.9% periodic acid (Carl Roth, Karslruhe, Germany), Schiff reagent (Merck, Darmstadt, Germany) and Mayer’s haematoxylin (Merck) the sections were dehydrated in an ascending ethanol series and xylene, and covered with HistoMount (National Diagnostics, Atlanta, USA). A NanoZoomer S360 (Hamamatsu, Geldern, Germany) was used to acquire images. To determine the extend of glomerular damage, the number of crescentic glomeruli was counted in a blinded manner. Crescents were defined as two or more cellular layers in the Bowman`s space. A minimum of 50 glomeruli were assessed per kidney^{18 (link)}.

Glycogen staining of the liver was performed using the periodic acid-Schiff reaction (PAS). The tissue slices were deparaffinised and rehydrated using xylol followed by a descending alcohol series [2 × 100%, 1 × 96%, 1 × 70%, 1 × aqua destillata (aqua dest.)]. The slices were oxidized in periodic acid (Carl Roth, Karlsruhe, Germany) for 15 min at room temperature, washed in aqua dest. for 5 min and stained for 15 min with Schiff reagent (Merck, Darmstadt, Germany) at room temperature. Counterstaining was performed with 25% hematoxylin for 10 s.

For histological analysis, tissue was cut into 1-μm-thick sections and deparaffinized by xylene treatment and rehydration in graded ethanol. Sections were stained with 0.9% periodic acid (cat# 3257.1, Roth) and Schiffsches Reagent (cat#1.09033, Merck) both for 10 min embedded into washing steps with H₂O. Finally, to visualize nuclei in blue, the samples were stained with Mayer’s Haematoxylin for 20 s. After dehydration of the sections, they were embedded with Histomount (HS-103, National Diagnostics). The DeadEnd™ Fluorometric TUNEL System (Promega) was performed following the manufacturer’s instructions, with the exception that the samples were mounted, with a pre-incubation of Hoechst (ThermoFisher Scientific, 1:1000) as nuclear staining, with ProLong™ Diamond (ThermoFisher Scientific). The antibody signals were visualized by using the Axio Observer as described above.