Molecular Analysis of Drosophila Milk Glands

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).

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Benoit J.B., Hansen I.A., Attardo G.M., Michalková V., Mireji P.O., Bargul J.L., Drake L.L., Masiga D.K, & Aksoy S. (2014). Aquaporins Are Critical for Provision of Water during Lactation and Intrauterine Progeny Hydration to Maintain Tsetse Fly Reproductive Success. PLoS Neglected Tropical Diseases, 8(4), e2517.

Publication 2014

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

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

Corresponding Organization : International Centre of Insect Physiology and Ecology

Top 5 similar protocols

Variable analysis

independent variables

Mated female flies with third instar larvae

dependent variables

Gene expression patterns in milk gland tubules

control variables

Fixation of milk gland tubules in Carnoy's fixative for 5 days
Preparation of samples according to Attardo et al. [47]
Use of Digoxigenin-labeled RNA probes generated using the MAXIscript T7 transcription kit
Use of anti-Digoxigenin-rhodamine Fab fragments (1:200 dilution) for FISH probe detection
Use of rabbit anti-gmmMGP (1:2500) antibodies
Use of Alexa Fluor 488 goat anti-rabbit IgG (1:500) as a secondary antibody for immunohistochemistry
Mounting of slides using VECTA SHIELD Mounting Medium with DAPI
Observation of samples using a Zeiss Axioskop2 microscope at 400x magnification
Imaging of samples using an Infinity1 USB 2.0 camera and software

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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