Anti digoxigenin rhodamine fab fragments for fish probe detection

Milk gland tubules together with fat body were collected from female flies carrying third instar larva and placed into Carnoy's fixative for a five day fixation period [53] (link). Digoxigenin-labeled RNA probes were generated using the MAXIscript T7 transcription kit following manufacturer's protocol (Ambion, Austin, TX) using a primer set with a T7 primer (Table S1) [52] . Antibody solutions were made featuring anti-Digoxigenin-rhodamine Fab fragments for FISH probe detection (1∶200 dilution) (Roche) and rabbit anti-GmmMGP (1∶2500) antibodies [39] (link), [53] (link). Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) at a dilution of 1∶500 was added as a secondary antibody for immunohistochemistry [53] (link). Slides were mounted in VECTASHIELD Mounting Medium with DAPI (Vector laboratories Inc. Burlingame, CA). Samples were observed using a Zeiss Axioskop2 microscope (Zeiss, Thornwood, NY) equipped with a fluorescent filter. Samples were viewed and imaged at 400× magnification. Images were captured using an Infinity1 USB 2.0 camera and software (Lumenera Corporation, Ottawa, Ontario, Canada).

Milk gland tubules were collected from mated female flies with third instar larvae and placed directly into Carnoy's fixative for a five day fixation period [47] (link). Samples were prepared according to Attardo et al. [47] (link) using Digoxigenin-labeled RNA probes generated using the MAXIscript T7 transcription kit following manufacturer's protocol (Ambion, Austin, TX) using a primer set with a T7 reverse primer (Table S1). Antibody solutions were made featuring anti-Digoxigenin-rhodamine Fab fragments for FISH probe detection (1∶200 dilution) (Roche) and rabbit anti-gmmMGP (1∶2500) antibodies [17] (link), [47] (link). Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) at a dilution of 1∶500 was added as a secondary antibody for immunohistochemistry [47] (link). Slides were mounted using VECTA SHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). Samples were observed using a Zeiss Axioskop2 microscope (Zeiss, Thornwood, NY) equipped with a fluorescent filter. Samples were viewed and imaged at 400× magnification. Images were captured using an Infinity1 USB 2.0 camera and software (Lumenera Corporation, Ottawa, Ontario, Canada).