This protocol is extracted from research article:
A sperm peptide enhances long-term memory in female Drosophila
Sci Adv, Nov 20, 2019; DOI: 10.1126/sciadv.aax3432

VT057280-Gal4 female flies were crossed with UAS-mCD8::GFP males. Before dissection, whole F1 female flies (3 to 4 days after eclosion at 25°C) were fixed in 4% paraformaldehyde in PBT (phosphate-buffered saline containing 1% Triton X-100) overnight at 4°C. Brains were dissected in Ca2+-free Drosophila Ringer’s solution and fixed for 1 hour at room temperature in 4% paraformaldehyde in PBT. Samples were then rinsed three times for 20 min in PBT, blocked with 2% bovine serum albumin in PBT for 2 hours, and incubated with rabbit anti-GFP (1:400; Invitrogen Molecular Probes) and mouse anti-nc82 (1:100; Developmental Studies Hybridoma Bank) primary antibodies in the blocking solution overnight at 4°C. Brains were washed three times for 20 min in PBT and then incubated with anti-rabbit secondary antibody conjugated to Alexa Fluor 488 and anti-mouse Alexa Fluor 591 (1:400; Invitrogen Molecular Probes) in the blocking solution overnight. After three washes (20 min), brains were mounted in ProLong Mounting Medium (Life Technologies) for microscopy analysis.

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