40 6 diamidino 2 phenylendole dapi

Whole larvae were fixed in paraformaldehyde at 4°C for 48 h, cryoprotected in 30% sucrose and mounted in O.C.T.™ medium (Sakura, Tissue-Tek, Alphen aan den Rijn, The Netherlands). Larvae were transversely sectioned in 10 μm thick slices using a cryostat (Leica, Wetzlar, Germany) at −20°C and were mounted on glass slides. Cryosections were blocked with a solution containing 0.1% Triton X-100 in phosphate-buffered saline (PBS) with 5% Horse serum for 30 min at room temperature. They were subsequently incubated at 4°C overnight with the following primary antibodies: mouse anti-Rho4d2 (1:7000; ab98887, Abcam, Cambridge, UK) and mouse anti-Zpr-1 (1:500; ab174435, Abcam). After several washes, sections were incubated with specific secondary antibodies: Cy3-conjugated anti-mouse IgG (1:800; 715-165-150, Jackson ImmunoResearch, West Grove, PA, USA), Cy3-conjugated anti-rabbit IgG (1:1000; 711-166-152, Jackson ImmunoResearch) or Alexa Fluor-488 conjugated anti-mouse IgG (1:1000; 715–545-150, Jackson ImmunoResearch). Nuclei were counterstained with 40,6-diamidino-2 phenylendole (DAPI; 1:5000; Sigma Aldrich). The emitted fluorescence was measured using a confocal microscope (LSM880 Fast Airyscan, Carl Zeiss, Jena, Germany).

Whole larvae were fixed in paraformaldehyde at 4°C for 48 h, cryoprotected in 30% sucrose and mounted in O.C.T. medium (Sakura, Tissue-Tek, Alphen aan den Rijn, the Netherlands). Larvae were transversely sectioned in 10-μm-thick slices using a cryostat (Leica, Wetzlar, Germany) at −20°C and mounted on glass slides. Cryosections were blocked with a solution containing 0.1% phosphate buffered saline/Triton X-100 and 5% horse serum for 30 min at room temperature. They were subsequently incubated at 4°C overnight with the following primary antibodies: mouse anti-Rho4d2 (1:7,000; ab98887, Abcam, Cambridge, UK) and mouse anti-Zpr-1 (1:500; ab174435, Abcam). After several washes, sections were incubated with specific secondary antibodies: Cy3 conjugated anti-mouse antibody (1:800; 715-165-150, Jackson ImmunoResearch, West Grove, PA), Cy3-conjugated anti-rabbit antibody (1:1,000; 711-166-152, Jackson ImmunoResearch), or Alexa Fluor 488 conjugated anti-mouse antibody (1:1,000; 715-545-150, Jackson ImmunoResearch). Nuclei were counterstained with 40,6-diamidino-2 phenylendole (DAPI) (1:5,000; Sigma Aldrich). The emitted fluorescence was measured using a confocal microscope (LSM880 Fastairyscan, Carl Zeiss, Jena, Germany).

Whole larvae were fixed in paraformaldehyde at 4 °C for 48 h, cryoprotected in 30% sucrose and mounted in O.C.T.^TM medium (Sakura, Tissue-Tek, Alphen aan den Rijn, The Netherlands). They were transversely sectioned in 10-µm thick slices using a cryostat (Leica, Wetzlar, Germany) at −20 °C and mounted on a glass slide. Cryosections were blocked with a solution containing 0.1% PBS/Triton X-100 and 5% horse serum for 30 min at room temperature. They were subsequently incubated overnight at 4 °C with the following primary antibodies: mouse anti-Rho4d2 (1:7000; ab98887, Abcam, Cambridge, UK), mouse anti-ZPR-1 (1:500; ab174435, Abcam) or rabbit anti-cleaved caspase-3 (Asp175) (1:500, #9661, Cell Signaling Technology, Danvers, MA, USA). After several washes, sections were incubated with the following conjugated secondary antibodies: Cy3 anti-mouse (dilution 1:800; #715-165-150, Jackson ImmunoResearch Europe Ltd., Ely, UK), Cy3 anti-rabbit (1:1000; #711-166-152, Jackson ImmunoResearch) or Alexa Fluor 488 anti-mouse (1:1000; #715-545-150, Jackson ImmunoResearch). Nuclei were counterstained with 40,6-diamidino-2 phenylendole (DAPI; 1:5000; Sigma Aldrich). The emitted fluorescence was measured using a confocal microscope (LSM880 Fastairyscan, Zeiss, Germany).