Wandering 3rd instar larvae were collected from respective genetic crosses and eye imaginal discs were processed for immunostaining as previously described (Sudhakaran et al. 2014 (link)). Each immunostaining experiment was reproduced in at least three independent biological replicates: for each replicate, discs dissected from multiple larvae were individually examined.
Primary antibodies used were: rabbit anti-RFP (Invitrogen R103367,) at 1:1000, rabbit-anti-Rasputin (Aguilera-Gomez et al. 2017 (link)) 1:1000, rabbit-anti-Caprin (Papoulas et al. 2010 (link)) at 1:1000, rat-anti-Elav-7E8A10 (Developmental Studies Hybridoma Bank) 1:300 and chicken-anti-Ataxin-2 (Bakthavachalu et al. 2018 (link)) at 1:500. Secondary antibodies were used at 1:1000 dilution: Alexa Fluor®488 goat anti-Chicken (Invitrogen A11039), Alexa Fluor®488 goat anti-Rabbit (Invitrogen A11078), Alexa Fluor®488 goat anti-Rat (Invitrogen A11006), and Fluor®555 goat anti-Rabbit (Invitrogen A21428).
Prepared eye-imaginal discs were mounted in Vectashield Mounting Medium (Vector Labs, H-1000) on microscope slides and imaged on a Zeiss LSM880 confocal microscope.
Primary antibodies used were: rabbit anti-RFP (Invitrogen R103367,) at 1:1000, rabbit-anti-Rasputin (Aguilera-Gomez et al. 2017 (link)) 1:1000, rabbit-anti-Caprin (Papoulas et al. 2010 (link)) at 1:1000, rat-anti-Elav-7E8A10 (Developmental Studies Hybridoma Bank) 1:300 and chicken-anti-Ataxin-2 (Bakthavachalu et al. 2018 (link)) at 1:500. Secondary antibodies were used at 1:1000 dilution: Alexa Fluor®488 goat anti-Chicken (Invitrogen A11039), Alexa Fluor®488 goat anti-Rabbit (Invitrogen A11078), Alexa Fluor®488 goat anti-Rat (Invitrogen A11006), and Fluor®555 goat anti-Rabbit (Invitrogen A21428).
Prepared eye-imaginal discs were mounted in Vectashield Mounting Medium (Vector Labs, H-1000) on microscope slides and imaged on a Zeiss LSM880 confocal microscope.
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