Mouse anti dcc intracellular domain

This was performed according to the manufacturer's instructions (Olink Bioscience) with modifications [39 (link)]. Briefly, eye explant cultures grown in glass-bottom dishes were fixed, permeabilized and blocked exactly as for immunostaining. The pair of primary antibodies, either mouse anti-DCC intracellular domain (1 : 100, BD Biosciences) and rabbit anti-ESCRT-II (1 : 200) or the same anti-DCC and rabbit anti-RPL5 (1 : 100, Proteintech Europe cat. no. 15430-1-AP), were applied and incubated overnight at 4°C. The dishes were washed 4× 5 min in PBS/0.002% Triton X-100 followed by application of anti-rabbit (+) and anti-mouse (−) probes for 1 h at 37°C, ligase for 30 min at 37°C and the polymerase mix with green fluorescence for 100 min at 37°C. The samples were mounted in Duolink mounting media and imaged on the same day.

Rabbit anti-Xenopus laevis ESCRT-II polyclonal antibody was made in the Blower laboratory; other antibodies were obtained as follows: mouse anti-acetylated α-tubulin (Sigma cat. no. T6793), mouse anti-α-tubulin (Sigma cat. no. T6074), mouse anti-Rab4 (BD Biosciences cat. no. 610888), mouse anti-Rab5 (BD Biosciences cat. no. 610281), mouse anti-Rab7 (Sigma cat. no. R8779), mouse anti-Rab11 (BD Biosciences cat. no. 610656), rabbit anti-β-tubulin (Abcam cat. no. ab6046), mouse anti-DCC intracellular domain (BD Biosciences cat. no. 554223), goat anti-DCC extracellular domain (R&D Systems cat. no. AF844), rabbit anti-RPL5 (Proteintech Europe cat. no. 15430-1-AP).