Roper spinning disc eclipse ti inverted microscope

The procedure of long-term imaging of disc explants was based on the protocol published in ref. ^{72 (link)} with minor modifications. We slightly modified the composition of the culture medium by adding adenosine deaminase (ADA, 8.3 ng/ml final concentration, Roche 10102105001) as proposed by recent findings of ref. ^{73 (link)}. In our hands, the addition of ADA in particular improved the long-term culture of young disc explants. In contrast, the addition of juvenile hormone (Methoprene) as proposed by Strassburger et al. has not proven beneficial in our setting and we did not use it in our culture medium.
As described previously^{72 (link)}, 72hAEL old larvae were dissected in culture medium and explants were immobilised between a round coverslip and a porous filter membrane (Whatman cyclopore polycarbonate membranes; Sigma, WHA70602513) using double-sided tape as spacers (~50 µm thickness, 3 M Scotch ATG 904 Clear Transfer Tape, No. 909-3799 from RS Components). The coverslip containing the mounted explants was inserted in an Attofluor chamber (A7816, ThermoFisher) and filled with 1 ml of culture medium. Explants were imaged on a Nikon Roper spinning disc Eclipse Ti inverted microscope (controlled by Metamorph 7.8.4.0) using a 40×–1.25 N.A. water-immersion objective at 22 °C. Image stacks of 1 µm z-spacing were acquired in 10 min intervals for up to 12 h.