Eosin g solution

Paraffin embedded sections were first heated in an oven at 65°C for 1 h before undergoing several series of washes of xylene and ethanol to deparaffinize/rehydrate. After deparaffinization/rehydration, the slides were immersed in Hematoxylin solution (Merck) for 3 min. The slides were rinsed with water, and counterstained with 0.5% Eosin G-solution (Merck) for 5 min. After 30 s of rinsing in water, the slides were dehydrated in series of increasing ethanol and xylene concentrations. The coverslips were mounted on the slides with Entellan Neu mounting medium (Merck).

Cryosections were also used for hematoxylin and eosin (H&E) staining. After washing the slides with H₂O (30 dips), they were stained with Mayer’s hemalum solution (Merck, Kenilworth, NJ, USA) for 6 min. After a second washing step with 30 dips in H₂O, slides were dipped 10 times in 0.3% HCl/EtOH solution and 30 times in H₂O and then incubated for 2 min in 0.5% Eosin G solution (Merck, Kenilworth, NJ, USA). Slides were then washed with 30 dips in H₂O, followed by 30 dips in isopropanol and 30 dips in HistoChoice^® Clearing Agent (Merck, Kenilworth, NJ, USA). Finally, cryosections were covered with ROTI^®Histokitt (CarlRoth, Karlsruhe, Germany) and stored at 4 °C. Samples were analyzed using an Axio Observer Z1 confocal laser scanning microscope attached to an LSM700 (Carl Zeiss, Berlin, Germany) and a VS120 automated slide scanner (Olympus, EVIDENT Europe GmbH, Hamburg, Germany).

Paraffin sections of 8 µm were used for hematoxylin and eosin stainings, where hematoxylin stains the cell nuclei and eosin stains the extracellular matrix and cytoplasm. After 30 washing steps with H₂O, slides were re-incubated for 6 min in Mayer’s hemalum solution (Merck, Kenilworth, IL, USA) followed by another 30 washing steps in H₂O. The cryosections are dipped 10 times in a 0.3% HCl/EtOH solution, 30 times in H₂O and further incubated for 2 min in a 0.5% Eosin G solution (Merck, Kenilworth, IL, USA). Slides were then washed 30 times in H₂O, 30 times in isopropanol, and 30 times in HistoChoice^® Clearing Agent (Merck, Kenilworth, IL, USA). Finally, paraffin sections were covered with ROTI^®Histokitt (Carl Roth, Karlsruhe, Germany) and stored at 4 °C.