Drosophila ovary extraction, fixation and immunostaining protocol

Drosophila ovary extraction, fixation and immunostaining protocol

Wen Lu, Vladimir I. Gelfand

Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, United States, 60611

1. Young mated female Drosophila were fed with dry active yeast for 16–18 hours (overnight) to promote oogenesis.
2. Ovaries were dissected from the adult fly abdomen using #55 forceps and carefully teased apart with 27G1/2 syringe needles in 1X Brinkley Renaturing Buffer 80 (1X BRB80: 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8) in a glass-bottom well.
   【Note: BRB80 buffer is crucial for maintaining microtubule integrity. Exposing the egg chambers from ovary bundles enhances extraction efficiency. 】
3. Dissected samples were extracted in extraction buffer (1X BRB80 + 1% Triton X-100) for 20 minutes without agitation. The original 1X BRB80 was gently replaced with the extraction buffer in the dissection glass well.
4. Extracted samples were carefully transferred into an Eppendorf tube using a large-opening pipette and fixed with 8% EM-grade formaldehyde in 1X BRB80 with 0.1% Triton X-100 for 20 minutes on a rotator.
   【Note: Handle extracted samples gently as they are fragile. Let the samples sit undisturbed in the fixation solution for 2–3 minutes before rotating to preserve structural integrity. 】
5. Samples were washed five times with 1X PBTB (1X PBS + 0.1% Triton X-100 + 0.2% BSA), 10 minutes each time, on the rotator.
6. Samples were blocked with 5% Normal Goat Serum (NGS) in 1X PBTB for 1 hour on the rotator.
7. Samples were incubated with a FITC-conjugated β-tubulin antibody (ProteinTech, Cat# CL488-66240) at a 1:100 dilution at 4 °C overnight on the rotator. Important: Protect samples from light during this step.
   【Note: Using a fluorescently conjugated tubulin antibody enhances staining quality, particularly in mid-oogenesis egg chambers, compared to standard primary/secondary antibody staining. 】
8. Samples were washed five times with 1X PBTB, 10 minutes each time, on the rotator. Important: Protect samples from light during this step.
9. [Optional] Samples were stained with rhodamine-conjugated phalloidin (0.2 µg/ml) and DAPI (1 µg/mL) for 1 hour and then quickly washed three times with 1X PBTB. Important: Protect samples from light during this step.
10. Samples were mounted in MOWIOL mounting medium and kept in the dark at room temperature overnight to allow the medium to solidify.
11. Samples were imaged using a Nikon W1 spinning disk confocal microscope (Yokogawa CSU with a 50 µm pinhole size), equipped with a Photometrics Prime 95B sCMOS camera and a 40× 1.25 N.A. silicone oil lens, controlled by Nikon Elements software. Images were acquired in z-stacks at 0.3 µm steps and processed using the Richardson-Lucy iterative deconvolution algorithm provided by Nikon Elements.