Neg 50 tissue tek medium

In order to cut cross sections of the retina samples, they were fixed with 4% paraformaldehyde (PFA; Merck, Darmstadt, Germany) for 15 min. Afterwards, the explants were drained with 15% sucrose solution (Sigma-Aldrich) for 15 min and 30% sucrose solution for 30 min. Finally, the explants were embedded in NEG-50 Tissue Tek medium (Thermo Fisher Scientific, Waltham, MA, United States) and stored at −80°C. Subsequently, a microtome (Thermo Fisher Scientific) was used to prepare 10 μm cross sections. Three tissue sections were placed on a Histobond slide (Paul Marienfeld GmbH & Co., KG, Lauda-Königshofen, Germany) and air-dried at room temperature overnight. For histological analyses, all slides were fixed in ice-cold acetone for 10 min on the following day and stored at −80°C.

After eight days of cultivation, the neuroretina samples (n = 8/group) were fixed with 4% paraformaldehyde (PFA; Merck) for 15 min. Next, they were drained with 15% sucrose solution (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and 30% sucrose solution for 30 min. In the final step, samples were embedded in NEG-50 Tissue Tek medium (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80 °C. Then, samples were cut into 10 µm cross-sections via a microtome (Thermo Fisher Scientific, Waltham, MA, USA) and placed on Histobond slides (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany) and air-dried at room temperature overnight. On the following day, all slides were fixed in ice-cold acetone for 10 min and stored at −80°C until further processed. Neuroretina samples for RT-qPCR analysis (n = 6/group) were immediately frozen at −80 °C (Figure 1B). Eight samples per group were stained for histology, whereas six samples of each investigated group were analyzed with RT-qPCR.