When staining Drosophila imaginal discs, often a rough dissection is performed where the discs are exposed but remain attached to the body. Once stained, the discs of interest are isolated and mounted. The example protocol demonstrates a procedure for wing imaginal disc dissection and mounting.
This protocol is an excerpt from Morimoto and Tamori, Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia, J. Vis. Exp. (2017).
1. Dissection of Larvae
- Collect wandering third instar larvae with a wooden stick or blunt forceps, genotype them by appropriate fluorescent markers (e.g., EGFP) under a fluorescence stereoscopic microscope and place them in a dissection dish with PBS (phosphate buffered saline).
- Wash the larvae in PBS by pipetting with 2 mL plastic transfer pipettes.
- Pinch the center of the larva with one forceps and tear the body in half with the other forceps.
- Pinch the mouth hook of the anterior half with one forceps and push the mouth towards the body with the other forceps to turn the body inside out.
- Remove unnecessary materials such as salivary glands or fat bodies with forceps.
2. Fixation and Antibody Staining
- Transfer the dissected anterior half of the larval body (including imaginal wing discs) to a 1.5 mL plastic tube and fix in 1 mL of Fix solution (4% Formaldehyde in PBS) for 10 min at room temperature in the dark with gentle rotation.
CAUTION: Formaldehyde is toxic and has carcinogenic potential. Wear protective gloves and clothing to prevent skin contact.
NOTE: In this part, all steps take place on a nutator at room temperature in the dark unless otherwise noted.
- Remove the Fix solution and discard. Wash the tissues with 1 mL of PBT (0.3% Triton X-100 in PBS) three times for 15 min each.
- Remove the PBT and add 1 mL of PBTG (0.2% bovine serum albumin and 5% normal goat serum in PBT) for blocking and nutate 1 h at room temperature or overnight at 4 °C.
- Remove PBTG and add primary antibody solution appropriately diluted with PBTG (see Table of Materials) and nutate overnight at 4 °C.
- Remove the primary antibody solution and wash the tissues with 1 mL PBT three times for 15 min each.
- Remove PBT and add secondary antibody solution appropriately diluted with PBTG (1:400). Nutate for 2 h at room temperature or overnight at 4 °C.
- Remove secondary antibody solution and wash the tissues with 1 mL of PBT two times for 15 min each.
- To stain F-actin, remove PBT and add Phalloidin solution appropriately diluted in PBS (1:40). Then nutate for 20 min. Remove the Phalloidin solution and wash the tissues with 1 mL of PBT two times for 15 min each.
- To counterstain nuclear DNA, remove PBT and add DAPI (4', 6-diamidino-2-phenylindole) solution (0.5 µg/mL of DAPI in PBS). Then nutate 10 min.
CAUTION: DAPI has carcinogenic potential. Wear protective gloves and clothing to prevent skin contact.
- Remove DAPI solution and wash the tissues with 1 mL of PBT two times for 15 min each.
- Rinse once in 1 mL of PBS for 10 min at room temperature.
- Remove PBS and add 500 µL of 100 % glycerol as the pre-mounting medium.
3. Mounting onto Microscope Slides
- Place the stained tissues on a microscope slide using a 2 mL plastic transfer pipette.
- Transfer the tissues to drops of mounting medium on another microscope slide with forceps.
- Hold down the end of dissected tissue with one forceps and pull away brain and eye antennal discs with the other forceps.
NOTE: Keep the dissected brains to place them near the wing imaginal discs. The brains act as a platform preventing the coverslip from crushing the wing imaginal discs.
- To isolate the wing imaginal discs hold down the end of dissected tissue with one forceps and gently scratch the body wall and tear off the discs with the other forceps.
NOTE: If it is difficult to find wing imaginal discs, peel the trachea from the posterior to the anterior side. Wing imaginal discs stick to the trachea.
- Gently cover the imaginal discs with a coverslip and seal with nail polish; store at 4 °C.
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|Phosphate buffered saline (PBS)||Wako||162-19321|
|bovine serum albumin||Sigma||A7906|
|normal goat serum||Sigma||G6767|
|mounting medium, Vectashield||Vector Laboratories||H-1000|
|mouse-anti-Dlg 4F3||Developmental Studies Hybridoma Bank||4F3 anti-discs large, RRID:AB_528203||dilute in PBTG, 1:40|
|mouse-anti-MMP1||Developmental Studies Hybridoma Bank||3A6B4, RRID:AB_579780||3 mixed 1:1:1 and dilute in PBTG, 1:40|
|mouse-anti-MMP1||Developmental Studies Hybridoma Bank||3B8D12, RRID:AB_579781||3 mixed 1:1:1 and dilute in PBTG, 1:40|
|mouse-anti-MMP1||Developmental Studies Hybridoma Bank||5H7B11, RRID:AB_579779||3 mixed 1:1:1 and dilute in PBTG, 1:40|
|mouse-anti-atubulin||Developmental Studies Hybridoma Bank||AA4.3, RRID:AB_579793||dilute in PBTG, 1:100|
|Alexa Fluor 546 Phalloidin||Molecular probes||A22283||dilute in PBS, 1:40|
|goat anti-mouse IgG antibody, Alexa Fluor 546||Molecular probes||A11030||dilute in PBTG, 1:400|
|sd-Gal4||Bloomington Drosophila Stock Center||#8609||recombined with UAS-EGFP|
|upd-Gal4||Bloomington Drosophila Stock Center||#26796||recombined with UAS-EGFP|
|UAS-lgl-RNAi||Vienna Drosophila RNAi center||#51247|
|UAS-scrib-RNAi||Vienna Drosophila RNAi center||#105412|
|UAS-RasV12||Bloomington Drosophila Stock Center||#64196|
|UAS-Yki3SA||Bloomington Drosophila Stock Center||#28817|
|hsFLP||Bloomington Drosophila Stock Center||#6|
|Act>CD2>GAL4 (flip-out GAL4)||Bloomington Drosophila Stock Center||#4780||recombined with UAS-EGFP|
|UAS-EGFP||Bloomington Drosophila Stock Center||#5428||X chromosome|
|UAS-EGFP||Bloomington Drosophila Stock Center||#6658||third chromosome|
|UAS-Dicer2||Bloomington Drosophila Stock Center||#24650||second chromosome|
|UAS-Dicer2||Bloomington Drosophila Stock Center||#24651||third chromosome|
|vkg-GFP||Morin et al. 2001||GFP protein trap|