Paraformaldehyde (pfa)

At indicated points in time, cells were fixed in paraformaldehyde (paraformaldehyde, 4%, Carl Roth) and incubated in permeabilization and blocking buffer (0.25% Triton X 100 and 10% secondary antibody-matched serum in Dulbecco's phosphate-buffered saline (DPBS)). Thereafter, cells were incubated with primary antibodies overnight at 4 °C and with secondary antibodies for 2 h at room temperature in the dark (Additional file 1: Table S1). Cells were stained with DAPI (Invitrogen) before images were acquired using the Axio Observer Z1 fluorescence microscope equipped with AxioVision software (version 4.9.1.0) (both Carl Zeiss). Image-based quantification of CPC marker expression was performed in ImageJ (version 1.53a, NIH) using 20X images and the Cell Counter plugin.

Isolated eyes were fixed for 30 min in 4% paraformaldehyde (Carl Roth GmbH, Karlsruhe, Germany) dissolved in phosphate-buffered saline (PBS) and washed twice for five minutes in PBS. The eyeball was opened, and the retina separated, incised to obtain a four-leaf clover shape. For staining, the retina was incubated in blocking buffer (1% BSA, Sigma-Aldrich, Munich, Germany, #A7030, and 0.3% Triton X100, AppliChem, Darmstadt, Germany, #A4975, in PBS) for two hours at room temperature or at 4 °C overnight, washed with PBS, and incubated with biotinylated isolectin B4 (Vector Laboratories, Eching, Germany; B1205, 1:25) or the primary antibody against the endothelial cell-specific molecule 1 (ESM1) (R&D Systems, Minneapolis, USA; AF1999, 1:100) overnight at 4 °C. ESM1 is a marker particularly for tip cells in a developing vasculature. After repeated washing, the retina was incubated with streptavidin conjugated with Cy3 (SigmaAldrich, Munich, Germany; S6402, 1:200) or a secondary antibody conjugated with Alexa488 (Invitrogen Life technologies, Darmstadt, Germany; A-11055, 1:200). Stained retinas were mounted on glass slides and inspected by a fluorescent confocal microscope (Leica TCS SP8 HCS A, Leica Microsystems, Wetzlar, Germany).

The tissue samples were sectioned vertically (thickness: 5 µm) 90° to the gas plasma-treated side (facing up) on Superfrost plus slides (Thermo Fisher, Bremen, Germany) and stored at −80 °C until further staining. Prior to staining, the sections were fixed in 4% paraformaldehyde (Carl Roth, Karlsruhe, Germany) and washed three times for five minutes in phosphate-buffered saline (PBS). Cell membrane permeabilization was performed for two minutes using 0.25% Triton X-100 (Carl Roth, Karlsruhe, Germany) followed by three washing periods of five minutes in PBS. The TUNEL kit (Roche Diagnostics, Basel, Switzerland) was used for apoptosis detection as per the manufacturer’s instructions. The slides were incubated for one hour at 37 °C in a humidified chamber. The sections were washed three times in PBS followed by DAPI addition to counterstain nuclei. The slides were mounted with Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, Taufkirchen, Germany). Imaging and analysis were performed using a high-content imaging device (Operetta CLS; PerkinElmer, Hamburg, Germany) and its associated software (Harmony 4.9; PerkinElmer, Hamburg, Germany). Quantification was based on algorithm-driven object segmentation.