✓Collect, culture, and stage Drosophila pupae for developmental analysis
✓Perform precise dissection of pupal abdominal tissue
✓Execute fixation, antibody staining, and immunohistochemistry protocols
✓Prepare and image immunostained samples for microscopy
Protocol
Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.
Difficulty
intermediate
Total time
~4-6 hours per batch (including fixation and incubation times)
Model organism
Drosophila melanogaster
Biosafety
BSL-1
Steps
1
Collect, culture, and stage Drosophila pupae
Establish synchronized Drosophila pupal populations and monitor developmental stages to ensure appropriate timing for dissection and analysis.
▶ 01:19
2
Dissect staged pupal abdominal tissue
Carefully isolate pupal abdominal segments under a microscope using fine dissection tools to obtain clean tissue samples.
▶ 02:33
3
Clean, fix, and incubate primary antibodies
Wash dissected tissue, apply fixative, permeabilize, block, and incubate with primary antibodies overnight or as specified.
▶ 03:40
4
Incubate secondary antibodies and mount samples
Apply fluorescently-labeled secondary antibodies, wash thoroughly, mount on slides with anti-fade mounting medium, and seal for imaging.
▶ 05:38
5
Image and evaluate prepared immunostained samples
Acquire fluorescence microscopy images of stained pupal abdominal tissue to visualize antibody-labeled developmental structures.
▶ 07:24
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