Female flies were used for gut immunostaining in all experiments. The entire gastrointestinal tracts were dissected out and fixed in 1× PBS plus 8% EM-grade paraformaldehyde (Polysciences) for 2 h. Samples were washed and incubated with primary and secondary antibodies in a solution containing 1× PBS, 0.5% BSA, and 0.1% Triton X-100. The following primary antibodies were used: rabbit anti-Gish (Tan et al., 2010 (link)), 1:500; mouse anti-Delta (Developmental Studies Hybridoma Bank), 1:100; rabbit anti-lacZ (Cell Signaling Technology), 1:1,000; mouse anti-pH3 (Millipore), 1:2,000; goat anti-GFP (Abcam), 1:500; rabbit anti–cleaved caspase-3, 5A1E (Cell Signaling Technology), 1:1,000; and DRAQ5 (Cell Signaling Technology), 1:5,000. Alexa Fluor–conjugated secondary antibodies were used at 1:400 (Jackson ImmunoResearch and Invitrogen). Guts were mounted in 70% glycerol and imaged with an inverted confocal microscope (LSM 710; Zeiss) using 10×, 20×, and 40× oil objectives (imaging medium: Immersol 518F; Zeiss). Imaging acquisition was performed at room temperature with LSM Image Browser (Zeiss), and image processing was done in Adobe Photoshop CC.
Protocol detail
Drosophila Gastrointestinal Tract Immunostaining
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EM-grade paraformaldehyde
mouse anti-Delta
mouse anti-pH3
goat anti-GFP
DRAQ5
Alexa Fluor–conjugated secondary antibodies
LSM 710
Immersol 518F
LSM Image Browser
Antibodies
Caspase 3
Confocal microscope
Female
Flies
Glycerol
Goat
Guts
Hybridoma
Lacz
Mouse
Paraformaldehyde
Rabbit
Triton x 100
Corresponding Organization : The University of Texas Southwestern Medical Center
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Variable analysis
independent variables
- Female flies were used for gut immunostaining in all experiments
- Gut immunostaining
- Entire gastrointestinal tracts were dissected out
- Samples were fixed in 1× PBS plus 8% EM-grade paraformaldehyde for 2 h
- Samples were washed and incubated with primary and secondary antibodies in a solution containing 1× PBS, 0.5% BSA, and 0.1% Triton X-100
- The following primary antibodies were used: rabbit anti-Gish, mouse anti-Delta, rabbit anti-lacZ, mouse anti-pH3, goat anti-GFP, rabbit anti–cleaved caspase-3, and DRAQ5
- Alexa Fluor–conjugated secondary antibodies were used at 1:400
- Guts were mounted in 70% glycerol and imaged with an inverted confocal microscope (LSM 710; Zeiss) using 10×, 20×, and 40× oil objectives (imaging medium: Immersol 518F; Zeiss)
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