Immunostaining of S2 cells and squashed salivary gland polytene chromosomes was performed as described previously [31 (link), 32 (link)]. The primary mouse polyclonal α-dCNDP2 antibodies were used at dilution 1:1000 and detected by goat α-mouse IgG antibodies conjugated to Alexa Fluor 488 (1:500; Invitrogen, A-11001). IF images of S2 cells were made using Zeiss LSM 710 confocal microscope and an oil 100×/1.40 plan apo lens. IF images of polytene chromosomes were acquired with a Zeiss Axio Observer.Z1 fluorescence microscope equipped with an Axiocam 506 mono (D) camera using an oil 63×/1.40 plan apo lens. In both cases, ZEN 2012 software was used for image acquisition.
For detection of eGFP-tagged dCNDP2 fusion proteins, whole salivary glands were fixed in PBS containing 4% formaldehyde (Merck, 104003), washed 3 times for 5 min each with PBS containing 0.5% Triton X-100, stained for 30 min with 0.4 μg/ml DAPI dissolved in PBS and mounted in 50% Glycerol dissolved in PBS. IF images of whole-mounted glands were obtained on a Zeiss LSM 710 confocal microscope using a 20×/0.50 EC Plan-Neofluar lens. Optical sections were combined using the LSM Image Browser version 3.5 software (Zeiss).
For detection of eGFP-tagged dCNDP2 fusion proteins, whole salivary glands were fixed in PBS containing 4% formaldehyde (Merck, 104003), washed 3 times for 5 min each with PBS containing 0.5% Triton X-100, stained for 30 min with 0.4 μg/ml DAPI dissolved in PBS and mounted in 50% Glycerol dissolved in PBS. IF images of whole-mounted glands were obtained on a Zeiss LSM 710 confocal microscope using a 20×/0.50 EC Plan-Neofluar lens. Optical sections were combined using the LSM Image Browser version 3.5 software (Zeiss).
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