Fluorescent Labeling of Milk Gland Tubules

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).

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Michalkova V., Benoit J.B., Attardo G.M., Medlock J, & Aksoy S. (2014). Amelioration of Reproduction-Associated Oxidative Stress in a Viviparous Insect Is Critical to Prevent Reproductive Senescence. PLoS ONE, 9(4), e87554.

Publication 2014

MAXIscript T7 transcription kit anti-Digoxigenin-rhodamine Fab fragments for FISH probe detection Alexa Fluor 488 goat anti-rabbit IgG VECTASHIELD Mounting Medium with DAPI Zeiss Axioskop2 microscope

Alexa fluor 488 Anti igg Antibodies Antibody Austin Dapi Digoxigenin Dilution Fab fragments Fat body Female Fish Fixative Flies Goat Immunohistochemistry Larva Microscope Milk Primer Rabbit Rhodamine Rna probes Transcription Vector

Corresponding Organization :

Other organizations : Institute of Zoology of the Slovak Academy of Sciences, Slovak Academy of Sciences, Yale University, Oregon State University

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