Hoechst 33258

Spheroids in fibrin gel were fixed in 4% paraformaldehyde (+4°C, overnight), washed thrice in PBS, permeabilized by 0.2% Triton X-100 (in PBS) for 10 min, and blocked with 5% goat serum in PBS. Samples were then incubated (+4°C, overnight) in the mix of PBS+0.1% Tween-20+5% goat serum with primary antibodies against CD31 (ab119339; Abcam), fibronectin (MA5-11981; Abcam), CD34 (ab54208; Abcam), and vimentin (ab92547; Abcam). After three times washing with PBST (PBS+0,1% tween), samples were then incubated in 250 μl of a solution of secondary species-specific antibodies conjugated with fluorochromes Alexa Fluor 488 and Alexa Fluor 594 (ThermoScientific, United States). All antibodies were used at 1/500 dilution. The nuclei were stained with 2 μg/ml of intercalating dye Hoechst 33258 (Serva, Germany) or DAPI (Abcam). The preparations in the mounting medium were covered with coverslips and examined under LSM 880 laser confocal scanning microscope (ZEISS, Germany) in visible and UV light.

Primary hippocampal neurons plated on coverslips were rinsed with PBS and fixed with a prewarmed solution of 4% paraformaldehyde and 4% sucrose for 15 min at room temperature. After permeabilizing the cells in methanol for 6 min at −20 °C, blocking was performed with 10% normal goat serum in PBS for 1 h at room temperature. Next, cells were incubated with primary antibody (diluted in PBS containing 2% BSA, 0,1% Triton X-100) for overnight at 4 °C, which continued with secondary antibody incubation (diluted in PBS containing 2% BSA, 0,1% Triton X-100) for 1 h at room temperature. Finally, coverslips were treated with Hoechst 33258 (2 μg/ml, Serva, Heidelberg, Germany) for 5 min and mounted on glass slides. When required, slices were photobleached prior to immunocytochemistry by incubation with 5% hydrogen peroxide (Sigma-Aldrich, Munich, Germany) for 45 min under white light at room temperature. For immunostaining of 5mC, prior to the abovementioned immunocytochemistry protocol, coverslips were incubated in methanol for 6 min at −20 °C followed by 1 M HCl incubation for 2 h at RT. We used the following primary antibodies for immunocytochemistry in this study: HA-tag (Santa Cruz, sc805 (1:500)), 5-mC (Calbiochem, NA81 (1:500)).