Tissue culture chambers

The microstructure of the samples was analysed with a FV1000-IX81 confocal laser scanning microscope (Olympus, Tokio, Japan) with a He-Ne laser (excitation wavelength 633 nm, emission detection between 565–615 nm). For a visualization of the undisturbed gel structure a modified protocol from Hickisch et al. [34 (link)] was used. After inoculation, samples were mixed with an aqueous 0.001% (w/v) solution of Nile Blue A Perchlorate to a concentration of 5% (v/v) of dye solution per sample and fermented in tissue culture chambers (Sarstedt, Nuembrecht, Germany). The unfermented samples were mixed with dye to the same concentration and analysed directly. Microscopy was performed on the tissue culture chambers with 60× magnification.

The LC-3 expression levels were determined using an immunofluorescence analysis. Cells were seeded in an 8-well Tissue Culture Chambers (Sarstedt AG & Co, Germany). After treatment with MASM 60 μg/ml with or without 50 μM Chloroquine for 8 h, the cells were fixed with 4% (v/v) paraformaldehyde (in PBS) for 10 min. After fixation, the cells were permeabilized with cold methanol for 10 min at − 20 °C and blocked with 2% (v/v) fetal bovine serum (FBS) and 1% (v/v) Goat Serum in PBS for 1 h at room temperature. After blocking, the cells were incubated with primary LC-3 antibody (1:100 diluted in blocking buffer) at − 4 °C overnight and then incubated with FITC-conjugated anti-rabbit IgG secondary antibody at room temperature for 1 h. Coverslips are mounted with DAPI (Invitrogen) to stain the nuclei. Samples were visualized using Cell Observer Z1 (Carl Zeiss Microscope, Germany) and images acquired were analyzed using Zeiss Zen Blue software suite.

Isolated hippocampi (from E18 embryos) were triturated in 1xHBSS (Invitrogen) after digestion by papain and DNase for 10 min at 37°C (Worthington). Transfections were performed using the Amaxa nucleofector system following the manufacturer's manual. 5 × 10⁶ cells and 3 μg of DNA mix was used for each transfection. Lifeact‐GFP, Lifeact‐RFP, Centrin2‐RFP, tDimer, mMaroon1, PaGFP, PaGFP‐UtrCH, Lifeact‐mEos3.2, mEos3.2, actin‐mEos4b, Drebrin‐YFP, Drebrin S142D‐YFP, Cofilin S3E‐GFP, Cofilin‐S3E‐RFP, Centrin2‐KR, EB3‐mCherry, and EB3‐GFP, plasmids were used for hippocampal neuronal transfections. The final concentration for each plasmid was 1 μg, except for Lifeact‐GFP, Lifeact‐RFP, and tDimer. We used 0.5 μg of Lifeact‐GFP, Lifeact‐RFP, and 0.3 μg of tDimer plasmids. In some cases, empty pcDNA 3.1 was used to make up to 3 μg of DNA per each transfection mix as per the manufacturer recommendation. After electroporation, neurons were plated on poly‐l‐lysine coated glass coverslips (for immunostaining), on glass‐bottomed dishes (ibidi, for live imaging) or tissue culture chambers (Sarstedt, for live imaging) in Neurobasal/B27 medium (Invitrogen) and were maintained in culture for 24 h at 37°C with 5% CO₂ before use.

Isolated hippocampi (from E18 embryos) were triturated in 1xHBSS (Invitrogen) after digestion by papain and DNase for 10 min at 37 °C (Worthington). Transfections were performed using the Amaxa nucleoporation system following the manufacturer’s manual. For each transfection 5 × 10⁶ cells and 5 µg of DNA mix were used for transfecting either control siRNA or Drebrin siRNA, and 3 µg of DNA mix for each of the remaining transfections. After electroporation, neurons were plated on poly-L-lysine coated glass coverslips (for immunostaining), on glass-bottomed dishes (ibidi, for live imaging) or tissue culture chambers (Sarstedt, for live imaging) in Neurobasal/B27 medium (Invitrogen), maintained in culture for 4~48 hours at 37 °C with 5% CO₂ before use.